Imaging apparatus and methods

ABSTRACT

Methods, systems and apparatus for manipulating electromagnetic radiation such as laser beams. A method and apparatus for correcting magnification chromatic aberration utilises one or more dispersive lenses such that long wavelength components are magnified less than short wavelength components. A telecentric relay is preferred to achieve this aim. Further, the use of polarisers to block the undesired zeroth order components of diffraction emanating from acousto-optic deflectors (AODs) is disclosed. Furthermore, specific designs of AOD including narrow transducer AODs which produce a diverging acoustic wave and AODs having two transducers and a selection switch are disclosed. Further, the invention provides methods, systems and apparatus for allowing the wavelength of radiation to be changed, for providing a user selectable degree of compensation, for providing a scanning and/or a pointing system and for providing a compact system that does not require telecentric relays between adjacent acousto-optic deflectors.

This application is a continuation of U.S. patent application Ser. No.12/440,809 filed on Nov. 25, 2009, which is a 371 U.S. National Stage ofInternational Application No. PCT/GB2007/003455, filed Sep. 12, 2007which claims the benefit of United Kingdom Patent Application No. GB0617945.1, filed Sep. 12, 2006. The disclosures of the aboveapplications are incorporated herein by reference.

The present invention relates to apparatus and methods involving themanipulation of a beam of electromagnetic radiation, such as a laserbeam. More particularly, the invention relates to apparatus and methodswhich use a laser beam to image a target space, such as by selectivelyfocussing the laser beam in the target space, which may be a 2D plane ora 3D volume. Several independent improvements to the state of the artare disclosed.

The ability to steer and focus electromagnetic radiation, such as alaser beam, rapidly in three-dimensions is very attractive for severalapplications in biology, microfabrication and data storage.

Laser scanning confocal imaging is an important and widely used tool inbiology because it allows high contrast visualization of subcellularstructures and monitoring of physiological processes with fluorescenceindicators within living tissue by excluding contaminating of out-offocus light. Conventional confocal methods work best at relativelyshallow depths where light penetration is good and scattering isminimal. Unfortunately, conventional confocal imaging cannot be used toimage biological activity deep (>100 μm) within the living tissue.However, more recently, a new type of laser scanning confocal microscopyhas been developed that relies on non-linear multiphoton excitation toselectively activate fluorophores where the light intensity exceeds the2-photon threshold at the centre of the focal volume. Fluorescent lightis emitted in all directions by these fluorophores and is picked up by ahigh numerical aperture lens system and photomultipliers. As the focalspot is scanned through the tissue the light intensity emitted by thefluorophores varies according to the intensity of staining by thefluorescence indicators in that part of the tissue. Combining thephotomultiplier signal with the known position of the 2-photon focalvolume enables a 2D or 3D image of the fluorescence intensity within thetissue to be reconstructed. This technique, known as two-photonmicroscopy, allows imaging at much greater depth because of the longerexcitation wavelengths used for multiphoton excitation (wavelengths of700-1000 nm), which scatter less than those used in conventionalconfocal imaging, and because confocality arises intrinsically from theexcitation volume allowing all emitted photons to be used to constructthe image. These properties together with the low levels of photodamageachievable have made 2-photon imaging an extremely powerful method forexamining physiological processes at the cellular and subcellular levelsboth in vitro and in vivo.

Two-photon imaging has been particularly popular in neuroscience, as ithas allowed the dynamic properties of neuronal network activity to beimaged in intact brain tissue using calcium indicators. The spatialresolution of 2-photon microscopy is well suited to this task evenallowing the small synaptic connections between neurons to be resolved.Multiphoton excitation has also begun to be used to photolyse “cagedcompounds” that release neurotransmitters, allowing synaptic inputs ontoa cell to be mimicked. This technique is potentially very important forunderstanding synaptic integration and thus determining how individualneurons carry out low-level computations.

Conventional laser scanning microscopes have traditionally usedgalvanometer minors to scan a laser beam. Such galvanometer mirrors areconfigured to scan in the X-Y plane only. Focussing in the Z directionis achieved by moving the apparatus relative to the sample (for exampleby moving the objective lens closer to or further away from the sample).The use of galvanometer mirrors has an inherent disadvantage in that theminors necessarily have a mass and the speed at which the mirror can bemoved from one position to another is limited by inertia. In practicalterms, this means that it takes of the order of 200-300 μs to move amirror from one selected position to another selected position. In turn,this limits the number of spots upon which a laser beam can be focussedduring a given time frame.

The temporal resolution of the present state of the artgalvanometer-based two-photon imaging systems is one or two orders ofmagnitude too slow to accurately image signalling in a network ofneurons. In such neurons, the elementary signal event (actionpotentials) occurs on the millisecond time scale. Moreover, the signalsare spatially distributed in three-dimensions as they flow through theneural networks and building a 3-D stack of images usinggalvanometer-based technology takes minutes. Furthermore, galvanometersare too slow for studying synaptic integration in individual neuronsusing photolysis because the excitation beam needs to be moved to many(for example 30) sites within a millisecond in order to stimulatesynapses distributed over the dendritic tree. For example, assuming thatit takes 300 μs to move from one spot to another using a galvanometerminor and assuming a dwell time at each spot of 5 μs, it would take 9.15ms to image 30 sites. This is approximately 10 times too slow forcurrent needs.

One approach suggested in the prior art to overcome some of thedisadvantages is to use rapid acousto-optic deflectors (AODs) instead ofgalvanometers to steer the two-photon laser beam. The advantage of usingAODs is that they allow the laser beam to be moved much more rapidlyfrom point-to-point than in a galvanometer-based system (compare amovement time of 5-25 μs with AODs to 200-400 μs with galvanometers).This has several potential advantages. Firstly, images can be scannedrapidly. Secondly, multiple point measurements can be made with longdwell times at very high temporal resolution (e.g. using an AOD systemwith 15 μs movement time, 33 points can be simultaneously sampled at 1KHz sample rate with a 15 μs dwell time or in other words 33 points canbe monitored 1000 times per second). The use of AODs therefore allowsmore of the time to be devoted to collecting photons from the regions ofinterest rather than being taken up in moving the laser beam betweensites.

As well as deflecting the laser beam in the X, Y plane, the use of twoAODs per axis can, in principle, also be used to focus the laser beam inthe Z dimension. For example, Kaplan et al describe in “Acousto-OpticLens with Very Fast Focus Scanning” Optics Letters, Vol. 26, No. 14,Jul. 15, 2001, pp 1078-1080, the use of two or four AODs to focus alaser beam in the X and Z plane or anywhere in an X, Y and Z volume. Toachieve focussing in a 3D volume, two AODs for focussing in the X-Zplane are followed by two AODs for focussing in the Y-Z plane.

One particular problem associated with multi-photon AOD scanning isspatial and temporal dispersion. Multi-photon applications typicallyrequire ultra-short laser pulses, for example of the order of 100 fs.However, the shorter the pulse, the larger the spread of wavelengthsthat exist in the pulse. The limiting example is an infinitely shortpulse which has a completely flat frequency spectrum (i.e. a whitespectrum). A pulse of 100 fs typically has a full-width, half-maximumspectral width of approximately 10 nm. The angle by which an AODdeflects a laser beam is related to the wavelength of the laser beam.Longer wavelengths are deflected more than shorter wavelengths. Thus, aform of spatial dispersion, also known as chromatic aberration, occurswhen an ultra-short pulse is deflected by an AOD. When a laser pulse isdiffracted through an AOD and then brought to a focus, the low frequency(long wavelength) parts of the pulse are focussed to a differentposition than the high frequency (short wavelength) parts of the pulse.This causes the pulse to be focussed to a line rather than a spot, thelength of the line being related to the spectral width of the pulse.Further, different wavelengths of light travel at different speedsthrough the AOD which causes temporal dispersion, i.e. elongation of thepulse in time. These problems are described in “Compensation of Spatialand Temporal Dispersion for Acousto-Optic Multiphoton Laser-ScanningMicroscopy” by Iyer et al, Journal of Biomedical Optics, 8(3), July2003, pp 460-471.

These dispersion problems provide two system limitations—(i) they worsenthe spatial resolution of the system, and (ii) they limit the excitationenergy density that is achieved at the focus, thus reducing excitationefficiency.

Iyer et al propose the use of a diffraction grating matched to thecentral acoustic wavelength in order to relieve some of the spatialdispersion. However, this solution only corrects the chromaticaberration for a single spot in the image plane with the chromaticaberration steadily increasing as you move away from this spot in theimage plane. This effect is known as magnification chromatic aberrationbecause it can also be described by saying that the longer wavelengthcomponent of the image has greater spatial magnification than theshorter wavelength component.

Reddy & Saggau (“Fast Three-Dimensional Laser Scanning Scheme UsingAcousto-Optic Deflectors”, Journal of Biomedical Optics, 10(6),November/December 2005) and Salome et al (“Ultrafast Random-AccessScanning in Two-Photon Microscopy using Acousto-Optic Deflectors”,Journal of Neuroscience Methods, 154 (2006), pp 161-174) disclose asimilar result in which two AODs are used to correct for chromaticaberration at one line in the image field but with chromatic aberrationincreasing as the deflection diverges from the compensation line. Thisis illustrated in FIG. 4 c of Reddy & Saggau.

Thus, even with the best compensation systems disclosed in the priorart, chromatic aberration can be eliminated at one single point only inthe field of view. The consequent focussing of a laser pulse to a line,rather than a diffraction limited spot, reduces the number of resolvablespots in the image plane. In a 3D image space, the effect of chromaticaberration can be quantified by considering the Number of ResolvableDetection Volumes (NRDV) in the image field. The detection volume of a2-photon system is the volume of tissue or other excitable material thathas sufficient intensity to be above its fluorescence activationthreshold. As 2-photon processes are dependent on the square of opticalintensity this volume is always smaller than the conventional focalvolume of the point spread function of the focused laser beam. The priorcompensation systems show increasing chromatic aberration as one movesaway from the compensation point and thus the NRDV is much less than ifthe chromatic aberration were corrected for substantially the wholeimage space. Such prior compensation systems therefore do not give thedesired spatial resolution to be used effectively for many applications,such as most 3D random access applications (e.g. neuroscienceapplications).

For such applications it is highly desirable to be able to randomlyaddress any location in a volume of approximately 250×250×250 μm with aspatial resolution of 1×1×2 μm at a rate of 20-30 μs per point. Thiscorresponds to a number of resolvable detection volumes (NRDV) of 7.8million. Detailed modelling of the designs proposed to date has shownthat the prior art methods struggle to achieve an NRDV in excess of200,000. This is a factor of 40 below what is a desirable target forneuroscience applications.

Reddy & Saggau disclose coupling two adjacent AODs using telecentricrelay optics (for example see FIG. 1 c of their paper, supra). When fourAODs are provided in a system capable of scanning a spot within a 3Dvolume, this necessitates at least three sets of telecentric relayoptics. A typical length of a prior art telecentric relay, in thedirection of the laser beam, is 400 mm. Thus, the requirement to utilisethree telecentric relays adds 1.2 m to the length of the system in thepath of the electromagnetic radiation. Accordingly, it is difficult toconstruct a device with a compact configuration and with minimal losses.It would be desirable to implement a shorter configuration, with lessloss-introducing components, but maintaining the functionality of theAODs in focussing the laser beam.

When an AOD deflects a laser beam, it is the first order component thatis usually of interest. The AOD will typically also pass an undeflectedzeroth order component that can interfere with the signal. Kaplan et alsolves this problem by ensuring that the two AODs deflect the laser beamin the same direction such that the undeflected zeroth order componentdoes not reach the image field. An alternative configuration proposed byReddy & Saggau of two parallel AODs suffers the potential problem thatundeflected zeroth order components can reach the image field. Note thatfor the highest efficiency, the anisotropic crystals of the deflectorsare used in the shear acoustic mode. These AODs have the property thatthe polarisation of the diffracted first order beam has its polarisationrotated through 90 degrees compared to the incoming laser beam and thezero order undeflected transmitted beam.

Another problem lies in the physical design of the AODs. Usually, theAOD devices are designed to have a good transmission efficiency (e.g.approximately 80%), but only for a very narrow range of input acceptanceangles, (typically ±1.5 mrad). As the input acceptance angle varies,efficiency typically reduces. Thus, when two AODs are used in series,the second AOD will receive light at an angle defined by the deflectionangle of the first AOD. Where the first AOD deflects the beam by arelatively large angle (e.g. greater than 1.5 mrad), this can cause thediffraction efficiency of the second AOD to be very low. It is thereforebe desirable to design an AOD having higher efficiencies at a largerrange of input acceptance angles.

There also exists a problem in that, for some applications, obtainingefficient transmission of the laser power is paramount, whereas forothers, obtaining high spatial accuracy is paramount. For example, theuse of a two-photon system for photolysis requires a much higher laserpower than when used for imaging. Further, a reduced NRDV can betolerated in photolysis applications. It would therefore be desirable tohave a system in which the NRDV/power trade-off can be varied inaccordance with the application to which the system is put.

It is convenient to be able to perform two-photon microscopy orphotolysis at a selected wavelength. Typically, the useful wavelengthrange is 700 nm to 1000 nm. However, there is a problem in thatdiffractive optics inherently deflect by different amounts at differentwavelengths (due to the fact that the diffraction angle increases as thewavelength increases). Providing a system that can operate under a rangeof different wavelengths is therefore difficult and would be desirable.

Furthermore, it would be desirable to provide a system that can provideselectable chromatic aberration correction. For example, it would bedesirable to provide a system that can be configured easily to correctall the chromatic aberration in one X-Y plane. Such a system shouldpreferably also be capable of being configured to correct all chromaticaberration in the Z plane. Preferably, such configuration should be viasimple means such as moving lens systems, rather than by replacingcomponents.

It is also desirable to provide a system that can operate in more thanone mode. For example, a pointing mode in which a series ofpredetermined points can be visited sequentially is useful. Also, ascanning mode, in which the laser beam focus moves smoothly over thetarget is also useful. A system which can be easily switched betweenthese modes is therefore very desirable.

It is furthermore desirable to provide a system which can performscanning in a smooth fashion even though there are limits on the minimumand maximum frequencies that can be put through an acousto-opticdeflector. These limits traditionally mean that scanning has to bestopped when the limit is reached. A system that can overcome thisproblem would be extremely desirable.

These and other problems are addressed by embodiments of the presentinvention.

In a first aspect, the present invention provides a system forselectively focussing a laser beam, said system comprising: diffractiveoptics for focussing the laser beam in an image field, said diffractiveoptics being such that, when said laser beam has spectral width, saiddiffractive optics will cause, in use, magnification chromaticaberration in said image field; and at least one optical element for atleast partially correcting said magnification chromatic aberration,which at least one optical element is arranged to modify said imagefield such that the longer wavelength components are magnified less thanthe shorter wavelength components.

The diffractive optics for deflecting and/or focussing the laser beamare preferably one or more acousto-optic deflectors.

The spectral width of the laser beam (for example 10 nm for a 100 fspulsed laser beam) causes chromatic aberration in the image field.Further there are different amounts of chromatic aberration at differentpositions in the image field. In the usual case, there is one point inthe image field, known as the compensation point, where chromaticaberration is at a minimum. The chromatic aberration generally increasesat positions away from this compensation point in the image field. Ifone takes a slice through the image field intersecting the compensationpoint, a graph of the chromatic aberration would cross the zero line atthe compensation point as the aberration changed from negative topositive (or vice versa) and would thus increase in magnitude eitherside of the compensation point, usually in a straight line. The gradientof this line is known as the magnification chromatic aberration and thefirst aspect of the invention diminishes the magnification chromaticaberration by reducing the gradient of this line. Thus, the magnitude ofthe chromatic aberration existing at every point in the image field isreduced (except at the compensation point where the magnitude is alreadyzero).

The system preferably comprises a laser for supplying the laser beam,which laser is preferably a pulsed laser having laser pulses of 2 ps orless, preferably 500 fs or less, more preferably still about 100 fs.

The centre frequency of the laser beam is typically in the range 600 to1000 nm, preferably 700 to 900 nm, more preferably 800 to 875 nm, andmore preferably still approximately 850 nm.

The correction can be carried out in a 2D image plane in which case itis possible to use either a modified microscope objective lens as theoptical element for correcting the magnification chromatic aberration oran additional dispersive lens prior to the objective, for instance atthe usual tube lens position or at some similar position earlier in theoptical relay chain.

In one embodiment, a telecentric relay is used to provide the necessarycorrection. Preferably, the telecentric relay has first and secondlenses and the rates of change of focal length with wavelength for thefirst and second lenses are of opposite sign. It is preferable that therates of change of focal length with wavelength for the first and secondlenses are of substantially the same magnitude. Preferably, the firstlens (i.e. the one the laser beam encounters first) has a shorter focallength for longer wavelengths than for shorter wavelengths and thesecond lens has a longer focal length for longer wavelengths than forshorter wavelengths. This means that the longer wavelengths aremagnified less by the telecentric relay and this provides the necessarycorrection to the magnification chromatic aberration.

The first and/or second lenses are preferably dispersive lenses and canbe made from combinations of crown glass, flint glass and diffractiveoptical elements.

The use of a telecentric relay allows the magnification chromaticaberration to be at least partially corrected for all points in a 3Dimage field and not just in a 2D plane.

In one preferable embodiment, a compensation factor C can be defined, avalue of C=1 providing perfect compensation for all of the chromaticaberration in the Z-direction, a value of C=2 providing perfectcompensation for all of the chromatic aberration in the X and Ydirections, and wherein C is selected to be less than 2.

Preferably, C is selected to be around 1.3.

In connection with the first aspect of the invention, there is alsoprovided a method for at least partially correcting magnificationchromatic aberration introduced into a laser beam by diffractive optics,said method comprising: passing said laser beam through at least oneoptical element so as to at least partially correct the magnificationchromatic aberration.

The diffractive optics can be one or more AODs or any alternativedynamically controlled system for deflecting and focussing a laser beam.Such alternative dynamic diffractive systems might for example be basedon liquid crystal holographic optical elements, magneto-optic arrays,digital micromirror arrays or any other spatial light modulator device.

In connection with the first aspect of the invention, there is alsoprovided a magnification chromatic aberration correcting telecentricrelay comprising: a first lens; second lens; wherein the rates of changeof focal length with wavelength for the first and second lenses are ofopposite sign in the wavelength range of interest.

Preferably, the rates of change of focal length of wavelengths for thefirst and second lenses are of substantially the same magnitude. Also,the first and second lenses can be separated by a distance approximatelyequal to the sum of their focal length for all wavelengths in thewavelength range of interest.

The lenses can be made of combinations of crown and flint glass. Morepreferably, the lenses comprise diffractive elements attached toconventional lenses.

A second aspect of the invention provides apparatus for selectivelydeflecting a laser beam, said apparatus comprising: a firstacousto-optic deflector that is arranged to modulate a laser beam intoat least (i) a zeroth order component of identical polarisation to theinput laser beam and (ii) a first order component having a polarisationrotated by 90° compared to the input laser beam; a first half-wave platethat is arranged to rotate the polarisation of the output of said firstacousto-optic deflector by 90°; a first polariser that is arranged topass said polarisation-rotated first order component and to block saidpolarisation-rotated zeroth order component; a second acousto-opticdeflector that is arranged to modulate said passed first order componentto produce at least (i) a second zeroth order component of identicalpolarisation to said passed first order component and (ii) a secondfirst order component of polarisation rotated by 90° compared to saidpassed first order component; a second polariser that is arranged topass said second first order component and to block said second zerothorder component.

The use of the half-wave plate and two polarisers allows the undesirablezeroth order components to be blocked effectively without substantiallyreducing the power of the desirable first order components.

The first and second acousto-optic deflectors can be used to deflect andfocus the laser beam in the X-Z plane. Additional third and fourthacousto-optic deflectors may be provided to provide additional focussingin the Y-Z plane. An additional half-wave plate and a further twopolarisers can be used to block the zeroth order components that may becreated in the third and fourth acousto-optic deflectors.

This construction allows the wanted output beam (that is the first orderdiffracted beam from each AOD) to be diffracted first in one direction,then in the opposite direction by the counter propagating acoustic wavein the second crystal of each pair. Thus the final net beam deflectionat the centre of the image field is zero. This makes the arrangementnaturally self compensating for chromatic dispersion at the centre ofthe image field. The use of the polarisers has eliminated thepotentially interfering zero order undeflected beams from each AOD.

In connection with the second aspect, there is also provided apparatusfor selectively deflecting a laser beam, said apparatus comprising: afirst acousto-optic deflector; a second acousto-optic deflector; a firstpolariser; and a second polariser.

The first polariser is preferably located between the first and secondacousto-optic deflectors. This allows it to cut out the unwanted zerodiffraction order and transmit the useful first diffraction order.

The first and second acousto-optic deflectors are conveniently arrangedto deflect and focus a laser beam in a first plane, such as in the X-Zplane, and the first and second polarisers are conveniently arranged topass only the first order components of diffraction (and block thezeroth order components of diffraction).

The first polariser preferably follows the first acousto-optic deflectorand the second polariser preferably follows the second acousto-opticdeflector.

To achieve additional deflection and focussing in the Y-Z plane, thirdand fourth acousto-optic deflectors can be provided together with thirdand fourth polarisers.

A particularly preferred configuration arranges the acousto-opticdeflectors in the order first, third, second, fourth. This dispenseswith the need for half-wave plates. Alternatively, the acousto-opticdeflectors can be arranged in the order first, second, third, fourth andtwo half-wave plates can be arranged between the first and second andthe third and fourth acousto-optic deflectors respectively.

In accordance with the second aspect, there is also provided a methodfor selectively deflecting a laser beam, said method comprising: usingfirst and second acousto-optic deflectors to focus a laser beam in afirst plane; and using first and second polarisers to pass the firstorder components of diffraction and to block any zeroth order componentsof diffraction.

Third and fourth acousto-optic deflectors are preferably used to focusthe laser beam in the second plane and third and fourth polarisers arepreferably used to pass the first order components of diffraction andblock any zeroth order components of diffraction.

The polariser of the laser beam is preferably rotated by 90° subsequentto the laser beam exiting the first and third deflectors but prior tothe laser beam entering the second and fourth polarisers respectively.

In accordance with a third aspect of the invention, there is providedapparatus for deflecting a laser beam, said apparatus comprising: afirst acousto-optic deflector optimised for efficient transmission atthe input laser beam angle; and a second acousto-optic deflector oflower peak efficiency than the first acousto-optic deflector but whichaccepts laser beams from a wider range of angles at better transmissionefficiency than said first acousto-optic deflector.

This aspect of the invention addresses the problem that, in the priorart, the range of angles at which a laser beam could enter the secondacousto-optic deflector of each X-Z or Y-Z pair is large which meansthat transmission efficiency is very poor at some input angles. Inaccordance with this aspect of the invention, the second acousto-opticdeflector is designed so as to have an efficiency versus input anglecurve having a flatter and broader peak than the first acousto-opticdeflector. Thus, although efficiency is reduced at the optimum inputangle, efficiency is increased at variations from the optimum angle suchas to provide acceptable transmission throughout the whole range ofpossible input angles. To maintain sufficient output power from thesystem for 2-photon imaging great care needs to be taken to minimiseother system losses. The loss of power can also be compensated byincreasing the power of the source laser.

There thus exists an optimum designed acceptance angle for the second ofeach pair of AODs (i.e. the second and fourth AODs). As their acceptanceangle increases so the NRDV increases as the scannable volume increases,but suddenly if the acceptance angle is increased beyond the optimum,the diffraction efficiency drops too low and the intensity of the laserspot reduces below the threshold required for 2-photon fluorescence andthe NRDV drops rapidly (note the NRDV only counts detection volumeswhere the laser intensity is above the two-photon threshold). Examplesof such optimisation curves can be seen in FIGS. 23 and 24.

The third aspect of the invention also provides a method of deflecting alaser beam, said method comprising: passing a beam through a firstacousto-optic deflector that has been optimised for efficienttransmission at the input laser beam angle; deflecting said beam usingsaid first acousto-optic deflector; passing said deflected beam througha second acousto-optic deflector that has a lower peak efficiency thansaid first acousto-optic deflector but which accepts laser beams from awider range of angles at better transmission efficiency that said firstacousto-optic deflector; and deflecting said beam using said secondacousto-optic deflector.

In a fourth aspect of the invention, there is provided apparatus fordeflecting a laser beam, said apparatus comprising: first and secondacousto-optic deflectors for focusing a laser beam in a first direction;and third and fourth acousto-optic deflectors for focusing a laser beamin a second direction; wherein said acousto-optic deflectors arearranged in this order along the path of the laser beam: first, third,second, fourth.

This particular order of acousto-optic deflectors dispenses with theneed for half-wave plates to rotate the polarisation of the light. Thisfirst acousto-optic deflector will transmit a first order component ofdiffraction that is rotated by 90° compared to the input laser beam. Thethird acousto-optic deflector is well-suited for receiving this firstorder component of diffraction and will transmit a further first ordercomponent that is rotated by a further 90°. The second acousto-opticdeflector is well-suited for receiving this laser beam and will againrotate the polarisation by a further 90°, making it suitable forreception by the fourth acousto-optic deflector. The inherentpolarisation rotation introduced to the first order components ofdiffraction by the AODs are, when this order of AODs is used, compatiblewith the polarisation acceptance of the next AOD in the sequence.

In accordance with the fourth aspect, there is also provided a method ofdeflecting a laser beam, said method comprising: using first and secondacousto-optic deflectors to focus a laser beam in a first direction; andusing second and third acousto-optic deflectors to focus a laser beam ina second direction; wherein said acousto-optic deflectors are arrangedin this order along the path of the laser beam: first, third, secondfourth.

A fifth aspect of the invention provides an acousto-optic deflectorcomprising: a crystal for propagating an acoustic wave that willdiffract an input laser beam; a first crystal transducer for supplyingacoustic vibrations to the crystal; and a second crystal transducer forsupplying acoustic vibrations to the crystal; wherein said first andsecond crystal transducers are located on the same side of the crystal.

The first crystal transducer is preferably arranged to create a morediverging acoustic wave in the crystal than said second crystaltransducer.

The effect of the more diverging acoustic wave is preferably to allowthe efficient diffraction of laser beams coming from a wider range ofangles.

A more diverging acoustic wave can be created by adjusting the width ofthe first crystal transducer to be smaller in the direction parallel tothe direction of light propagation. For example, a crystal transducerwidth of less than 1 mm can create an appropriate diverging acousticwave.

The second crystal transducer is preferably wider in the direction oflight propagation than the first crystal transducer. This allows thefirst crystal transducer to be one which supplies a more divergingacoustic wave and the second crystal transducer to be one which suppliesa less diverging acoustic wave. The two transducers therefore arepreferably designed to create acoustic waves having different propertiesgiving added flexibility to the system.

Preferably, each crystal transducer can be independently selectable suchthat one or both may be excited to modulate the divergence of theacoustic wave in the crystal.

There can be provided any number of crystals, such as one, two, three,four or more.

A switch mechanism can be provided to selectively allow for only onetransducer to be excited, two transducers to be excited together orthree transducers to be excited together. The transducers are preferablyadjacent to one another.

In a preferred embodiment, the width of each transducer increases in ageometric progression in a direction parallel to the direction of lightpropagation.

In accordance with a sixth aspect of the invention, and a selectionswitch is provided to allow either the first or second crystaltransducer to be excited. In more particularity, the sixth aspect of theinvention provides an acousto-optic deflector comprising: a crystal forpropagating an acoustic wave that will diffract an input laser beam; afirst crystal transducer for supplying acoustic vibrations to thecrystal; a second crystal transducer for supplying acoustic vibrationsto the crystal; and a selection switch for selecting whether the firstor second crystal transducer is excited.

The sixth aspect of the invention provides a method of deflecting alaser beam, said method comprising: selecting one of a first or secondcrystal transducer arranged to supply acoustic vibrations to a crystal;exciting said selected crystal transducer so as to propagate an acousticwave in said crystal; and diffracting said laser beam with said acousticwave.

In accordance with a seventh aspect of the invention, the crystal of theacousto-optic deflector has a particular orientation. More specifically,the seventh aspect of the invention provides an acousto-optic deflectorcomprising: a crystal having a laser input direction defined by thenegative Z-axis; wherein said crystal structure is rotated byapproximately 2° about the X-axis and approximately 3° about the Y-axis.

Also in accordance with the seventh aspect of the invention, there isprovided an acousto-optic deflector comprising: a crystal oriented suchthat acoustic waves propagating therethrough will have approximately 20°between their wave vector and their Poynting vector.

Preferably, the second of a pair of acousto-optic deflectors has aconstruction in accordance with the second aspect of the invention. Insuch a case, it is useful that the first deflector in the pair also hasthe same construction.

According to an eighth aspect of the invention, there are providedsystems and methods which can account for a non-zero effective opticalseparation between adjacent AODs.

This can preferably be achieved by providing a system for manipulating abeam of electromagnetic radiation, said system comprising: a firstacousto-optic deflector; a second acousto-optic deflector positioneddownstream of said first acousto-optic deflector and being separatedfrom said first acousto-optic deflector by an effective opticalseparation; a driver for providing acoustic waves in said first andsecond acousto-optic deflectors, said acoustic waves being chirped atdifferent ramp rates to account for said effective optical separationbetween said first and second acousto-optic deflectors.

Preferably, the driver is arranged to provide acoustic waves that causethe electromagnetic radiation to be focused to a stationary line inspace.

Preferably, there is provided a system wherein said driver provides anacoustic wave with a ramp rate a₁ to said first acousto-optic deflectorand provides an acoustic wave with a ramp rate a₂ to said secondacousto-optic deflector, and wherein said ramp rates are related by:

$\frac{a_{1}}{a_{2}} = \frac{2d_{2}^{\prime}}{{2d_{2}^{\prime}} + d_{1}}$

where d₁ is the effective optical separation between said first andsecond acousto-optic deflectors and d′₂ is the effective opticaldistance to the focal line from the second acousto-optic deflector.

In preferred embodiments, the system further comprises a thirdacousto-optic deflector; a fourth acousto-optic deflector positioneddownstream of said third acousto-optic deflector and being separatedfrom said third acousto-optic deflector by an effective opticalseparation; wherein said driver is arranged to provide acoustic waves insaid third and fourth acousto-optic deflectors, said acoustic wavesbeing chirped at different ramp rates to account for said effectiveoptical separation between said third and fourth acousto-opticdeflectors.

The driver is preferably arranged to select frequencies of the acousticwaves that scan a target in the X and/or Y direction.

The driver is preferably arranged to select frequencies for said firstand second acousto-optic deflectors such as to achieve an angular scanrate of δθ/δt by adjusting the ramp rate a₁ of the first acousto-opticdeflector to be:

$a_{1} = \frac{\frac{V}{\lambda}( {\frac{V}{2d_{2}^{\prime}} - \frac{\delta\theta}{\delta \; t}} )}{2 + \frac{d_{1}}{d_{2}^{\prime}} - {\frac{d_{1}}{V}\frac{\delta\theta}{\delta \; t}}}$

and by adjusting the ramp rate a₂ of the second acousto-optic deflectorto be:

$a_{2} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}$

where V is the speed of sound in the first and second acousto-opticdeflectors, λ is the wavelength of the laser beam to be deflected, d′₂is the distance to the focal line/point from the second acousto-opticdeflector and d₁ is the effective optical separation between said firstand second acousto-optic deflectors.

The driver preferably provides acoustic waves such as to scan a targetin the Z and/or Y direction, said scan being composed of a series ofmini-scans, with a non-active scan period between each active scan timeof each mini-scan.

This non-active period can be used to adjust the value of thefrequencies without moving the focus position and preferably consists ofa frequency resetting time and a AOD fill time.

The non-active time starts at the end of the active scan time of onemini-scan and ends at the beginning of the active scan time of thesubsequent mini-scan.

The active scan time is preferably that time for which measurements aretaken and, generally, measurements are not taken during the non-activetime duration.

Also included in the eighth aspect of the invention is a method ofmanipulating a beam of electromagnetic radiation, said method comprisingpassing said electromagnetic radiation through a first acousto-opticdeflector and a second acousto-optic deflector downstream of said firstacousto-optic deflector, the deflectors containing first and secondacoustic waves respectively; wherein said first and second acousticwaves are chirped at different ramp rates to account for the effectiveoptical separation between said first and second acousto-opticdeflectors.

In accordance with the ninth aspect of the invention, there is provideda method of scanning a target volume with a beam of electromagneticradiation, said method comprising passing said electromagnetic radiationthrough a first acousto-optic deflector and a second acousto-opticdeflector downstream of said first acousto-optic deflector, thedeflectors containing first and second acoustic waves respectively so asto move a focus position of said beam along a scan path in said targetvolume; wherein said first and second acoustic waves are chirped to havea constantly increasing or decreasing frequency; and when one of saidacoustic waves is at a predetermined maximum or minimum frequency value,offsetting the frequency of each acoustic wave such that the acousticwaves may continue to be chirped while having frequencies lower thansaid predetermined maximum frequency and higher than said predeterminedminimum frequency.

The frequency offsetting can be carried out whether or not theacousto-optic deflectors are telecentrically coupled or there is a realoptical separation between the deflectors. Preferably, said first andsecond acousto-optic deflectors are separated by an effective opticalseparation d₁ and said focal position is an effective optical distanced′₂ from said second acousto-optic deflector, said offsets satisfying:

$\frac{\Delta \; f_{1}}{\Delta \; f_{2}} = \frac{2d_{2}^{\prime}}{{2d_{2}^{\prime}} + d_{1}}$

where Δf₁ is the frequency offset for the first acoustic wave and Δf₂ isthe frequency offset for the second acoustic wave.

Preferably, the target volume is scanned as a series of mini-scans, theactive scan time of each mini-scan terminating approximately at thepoint where the frequency of each acoustic wave is offset and the activescan time of subsequent mini-scans beginning after a non-active periodfrom said termination point of the previous mini-scan.

Also in accordance with the ninth aspect of the invention there isprovided a system for scanning a target volume, said system comprising afirst acousto-optic deflector; a second acousto-optic deflectorpositioned downstream of said first acousto-optic deflector; a driverfor providing acoustic waves in said first and second acousto-opticdeflectors; wherein said driver is arranged to offset the frequency ofeach acoustic wave when one of said acoustic waves reaches apredetermined maximum or minimum frequency value so as to maintain apredetermined chirp rate for said acoustic weaves while keeping theabsolute frequency value for said first and second acoustic wavesbetween said predetermined minimum and maximum frequency values.

In accordance with the tenth aspect of the invention, there is provideda system for selectively focussing a beam of electromagnetic radiation,said system comprising diffractive optics for focussing the beam in animage field, said diffractive optics causing chromatic aberration insaid image field; corrective optics for at least partially correctingsaid chromatic aberration, said corrective optics being adjustable suchthat said chromatic aberration can be at least partially corrected whenthe electromagnetic radiation has a wavelength falling within a range ofwavelengths of interest.

Preferably, the corrective optics is capable of ensuring that the beamof electromagnetic radiation fills the same design system aperture forsubstantially all wavelengths falling within the range of the wavelengthof interest. For example, the radiation can be made to fill the apertureof the system objective lens to provide the maximum focussingresolution.

The corrective optics preferably comprises first and second diffractivecorrection plates.

These correction plates may also be comprised of conventional lenses.

Preferably, the first correction plate has a first diffractive elementhaving positive power attached to a negative focal length real lens andthe second correction plate has a second diffractive element havingnegative power, attached to a positive focal length real lens. Theattachment is preferably intimate, for example by optical gluing.

The diffractive optical element and real lens in each correction plateis preferably balanced such that at a predetermined wavelength in themid-operating range (for example 800-850 nm), both correction plates areof close to zero effective power.

Further lenses can be placed either side of the correction plate.

These further lenses can be implemented by a pair of zoom lenses.

In accordance with the tenth aspect, there is provided a method ofselectively focussing a beam of electromagnetic radiation, said methodcomprising passing said beam of electromagnetic radiation throughdiffractive optics to focus the beam in an image field, said diffractiveoptics causing chromatic aberration in said image field; adjustingcorrective optics when the wavelength of said electromagnetic radiationis changed within a range of wavelengths of interest; passing saidelectromagnetic radiation through said adjusted corrective optics to atleast partially correct said chromatic aberration.

In accordance with an eleventh aspect of the invention, there isprovided a system for selectively focussing a beam of electromagneticradiation, said system comprising diffractive optics for focussing thebeam in an image field, said diffractive optics causing chromaticaberration in said image field; corrective optics for at least partiallycorrecting said chromatic aberration, said corrective optics beingcapable of substantially fully correcting chromatic aberration in an X-Yplane when a compensation factor C is equal to 2 and being capable ofsubstantially fully correcting chromatic aberration in a Z plane whensaid compensation factor C is equal to 1, said compensation factor beinguser selectable.

The compensation factor C is preferably set by moving diffractiveelements.

The system is preferably arranged to receive from a user a desiredcompensation factor and a desired electromagnetic radiation wavelength.

Preferably, upon receiving such input, the system moves elements of thecorrective optics so as to provide a chromatic aberration correction inaccordance with the compensation factor at the desired wavelength.

In accordance with the eleventh aspect, there is provided a method ofselectively focussing a beam of electromagnetic radiation, said methodcomprising passing said electromagnetic radiation through diffractiveoptics, said diffractive optics causing chromatic aberration in an imagefield; selecting a compensation factor C; configuring corrective opticsin accordance with said selected compensation factor C; passing saidelectromagnetic radiation through said corrective optics to at leastpartially correct said chromatic aberration, wherein said correctiveoptics are capable of substantially fully correcting chromaticaberration in an X-Y plane when said compensation factor C is equal to 2and are capable of substantially fully correcting chromatic aberrationin a Z plane when said compensation factor C is equal to 1.

In accordance with the twelfth aspect of the invention, there isprovided a system for manipulating a beam of electromagnetic radiation,said apparatus comprising a first acousto-optic deflector; a secondacousto-optic deflector; a driver for providing first and secondacoustic waves to said first and second acousto-optic deflectorsrespectively; a user operated switch for selecting between a randomaccess mode and a scanning mode.

Preferably, when said random access mode is selected, a series of pointsin a target volume can be programmed into the system and theacousto-optic deflectors are thereafter used to focus a beam ofelectromagnetic radiation to each of said plurality of points in thetarget volume for a predetermined dwell time.

Preferably, when the scan mode is selected the system is arranged toscan a focal position along a predetermined path using saidacousto-optic deflectors.

Preferably, the scan is made up of a plurality mini-scans having aduration determined in part by the Z-position at which scanning takesplace.

Preferably, the apparatus is configured to perform the following method:scanning a target in three dimensions; presenting to a user images ofthe target; receiving inputs from the user identifying a plurality ofpoints within the target; calculating the signals to provide to saiddriver for causing the beam of electromagnetic radiation to sequentiallypoint to said selected plurality of points in the target; and pointingthe beam of electromagnetic radiation sequentially to said selectedpoints.

This aspect of the invention also provides a method of manipulating abeam of electromagnetic radiation, said method comprising determining aselection from a user; passing said beam of electromagnetic radiationthrough first and second acousto-optic deflectors, said first and secondacousto-optic deflectors respectively containing first and secondacoustic waves; wherein when a user has selected a random access mode,said waves are configured to cause said beam of electromagneticradiation to sequentially point to a series of points within athree-dimensional volume for a predetermined respective dwell time; andwhen said user has selected a scanning mode, said waves are configuredto cause said beam of electromagnetic radiation to scan a path in saidthree-dimensional volume at a predetermined scan rate.

In addition, this aspect can provide a method of manipulating a beam ofelectromagnetic radiation, said method comprising: scanning a beam ofelectromagnetic radiation around a path in a three-dimensional volume soas to provide an image of said volume; receiving an identification of aplurality of points within said target volume from a user; sequentiallypointing said beam of electromagnetic radiation to said plurality ofidentified points.

In accordance with a thirteenth aspect of the invention, there isprovided a method of scanning a target volume with a beam ofelectromagnetic radiation, said method comprising passing saidelectromagnetic radiation through a first acousto-optic deflector and asecond acousto-optic deflector downstream of said first acousto-opticdeflector, the deflectors containing first and second acoustic wavesrespectively so as to move a focus position of said beam along a scanpath in said target volume at an angular scan rate given by δθ/δt;wherein said first and second acoustic waves are chirped to have aconstantly increasing or decreasing frequency; and wherein the ramprates of said chirped acoustic waves are selected in accordance with:

$a_{1} = \frac{\frac{V}{\lambda}( {\frac{V}{d_{2}^{\prime}} - \frac{\delta\theta}{\delta \; t}} )}{2 + \frac{d_{1}}{d_{2}^{\prime}} - {\frac{d_{1}}{V}\frac{\delta\theta}{\delta \; t}}}$$a_{2} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}$

where a₁ is the ramp rate in the first acousto-optic deflector, a₂ isthe ramp rate in the second acousto-optic deflector, V is the speed ofsound in the first and second acousto-optic deflectors, λ is thewavelength of the electromagnetic radiation beam, d₁ is the effectiveoptical separation between the first and second acousto-optic deflectorsand d′₂ is the distance to the focus position from the secondacousto-optic deflector.

The value d₁ can be made zero, in which case the acousto-opticdeflectors can be coupled together by a telecentric relay.

Alternatively, the value of d₁ can be made non-zero and the values ofthe chirp rates can be found in accordance with the above equations(taking into account the small corrections to these equations that maybe needed to account for small errors in the alignment of components).

Preferably, the ramp rate a₂ of the acoustic wave in the secondacousto-optic deflector is determined such that the additional curvatureprovided to the wavefront of said electromagnetic radiation by saidsecond acousto-optic deflector is a predetermined amount more or lessthan the curvature of the wavefront as it arrives at said secondacousto-optic deflector from said first acousto-optic deflector, such asto provide for the scanning of said focal position.

In accordance with this aspect there is provided a system for scanning atarget volume with a beam of electromagnetic radiation, said systemcomprising: a first acousto-optic deflector; a second acousto-opticdeflector positioned downstream of said first acousto-optic deflectorand being separated from said first acousto-optic deflector by aneffective optical separation; a driver for providing respective firstand second acoustic waves in said first and second acousto-opticdeflectors, said first acoustic wave having a ramp rate given by;

$a_{1} = \frac{\frac{V}{\lambda}( {\frac{V}{d_{2}^{\prime}} - \frac{\delta\theta}{\delta \; t}} )}{2 + \frac{d_{1}}{d_{2}^{\prime}} - {\frac{d_{1}}{V}\frac{\delta\theta}{\delta \; t}}}$

and said second acoustic wave having a ramp rate given by:

$a_{2} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}$

where a₁ is the ramp rate in the first acousto-optic deflector, a₂ isthe ramp rate in the second acousto-optic deflector, V is the speed ofsound in the first and second acousto-optic deflectors, λ is thewavelength of the electromagnetic radiation beam, d₁ is the effectiveoptical separation between the first and second acousto-optic deflectorsand d′₂ is the distance to the focus position from the secondacousto-optic deflector.

In any of the aspects of the present invention, the following arepreferable features.

The electromagnetic radiation is selectively focussed to a line and/orto a point.

The electromagnetic radiation passes through a system comprisingmicroscope optics, for example a system including a microscope objectivelens.

The method, apparatus and system of the present invention isparticularly useful for implementing non-linear optical processes, suchas multi-photon processes or two-photon processes.

In all embodiments, chromatic aberration is preferably substantiallycorrected over a 3D image field.

Any of the acousto-optic deflectors of the present invention arepreferably made from a higher frequency anisotropic acousto-opticcrystal of which TeO₂ is one example.

The present invention will now be further described, by way ofnon-limitative example only, with reference to the accompanyingschematic drawings, in which:—

FIG. 1 shows an acousto-optic deflector (AOD) and the principle ofdiffraction of a laser beam using an ultrasonic acoustic wave;

FIG. 2 shows an AOD focussing a laser beam;

FIG. 3 shows the moving focal spot obtainable with a single AOD;

FIG. 4 a shows a graph of the frequency of the acoustic wave as itvaries with time;

FIG. 4 b shows a graph of the frequency of the acoustic wave as itvaries with distance across the AOD;

FIG. 5 shows a configuration comprising two AODs which allow a laserbeam to be focussed to a fixed spot in the X-Z plane;

FIG. 6 is a similar view to FIG. 5 but additionally shows theundiffracted zeroth order component of diffraction;

FIGS. 7 a-7 c show how a lens 70 can be used to focus the AOD output toa real position in a target;

FIG. 8 shows a configuration of two parallel AODs in accordance with thepresent invention;

FIG. 9 is similar to FIG. 8 and shows the chromatic aberration that canoccur when the input laser has a spectral width;

FIG. 10 shows an overview of the components of a two-photon systemaccording to the present invention;

FIGS. 11 a, 11 b and 11 c show graphs of the chromatic aberration versusthe distance X in the image field. FIG. 11 a shows the completelyuncorrected graph, FIG. 11 b shows a graph corrected at a single pointin the image field and FIG. 11 c shows a graph in which magnificationchromatic aberration has also been corrected;

FIG. 12 is a graph showing the chromatic aberration at various points inthe image field;

FIG. 13 is a graph similar to FIG. 12 showing the chromatic aberrationat various points in the image field;

FIG. 14 shows a system with a modified tube lens or microscope objectivelens in accordance with the present invention;

FIG. 15 shows a dispersive lens in accordance with the presentinvention;

FIG. 16 shows a telecentric relay in accordance with the presentinvention;

FIG. 17 shows the telecentric relay in accordance with the presentinvention together with an objective lens;

FIG. 18 shows a telecentric relay and objective lens in accordance withanother configuration of the present invention;

FIG. 19 shows a telecentric relay and objective lens in accordance witha further configuration of the present invention;

FIG. 20 is a graph showing the improvement in magnification chromaticaberration obtainable with the present invention;

FIG. 21 is a graph showing the number of resolvable detection volumes(NRDV) and how this varies with a correction factor C related to theamount of magnification that the shorter wavelengths are subjected tocompared to the longer wavelengths;

FIG. 22 is a graph similar to FIG. 20 in which the chromatic aberrationhas been corrected perfectly in the X-direction;

FIG. 23 is a graph similar to FIG. 20, in which the chromatic aberrationhas been corrected perfectly in the Z-direction;

FIG. 24 is a graph showing the number of resolvable detection volumes(NRDV) and how this varies with design acceptance angle range of thesecond AOD in an AOD pair according to the prior art;

FIG. 25 is a graph showing the number of resolvable detection volumes(NRDV) and how this varies with design acceptance angle range of thesecond AOD in an AOD pair according to the present invention;

FIG. 26 is an arrangement of two AODs according to the presentinvention;

FIG. 27 shows two orthogonal views of an arrangement of four AODs inaccordance with the present invention;

FIG. 28 is a three-dimensional plot of the diffraction efficiency of aknown AOD;

FIG. 29 is the same as FIG. 28, but viewed from a different direction;

FIG. 30 shows a plot of the diffraction efficiency of an AOD inaccordance with the present invention;

FIG. 31 shows an AOD crystal having a wide ultrasonic transducer;

FIG. 32 shows an AOD crystal having a narrow ultrasonic transducer;

FIG. 33 shows an AOD crystal having a selectable pair of transducers;

FIG. 34 shows an AOD crystal having three independently selectabletransducers;

FIGS. 35 a and 35 b show a practical arrangement of AODs and telecentricrelays;

FIG. 36 shows a laser beam being focussed by two AODs;

FIG. 37 shows the AOD drive frequencies utilised in the arrangement ofFIG. 36;

FIG. 38 shows distances used to derive equations to explain the ramprates used in the AOD configuration of FIG. 36;

FIGS. 39 a and 39 b show a configuration of four AODs;

FIG. 40 shows a scan pattern in the X-Y plane;

FIG. 41 shows raster scanning with four AODs;

FIG. 42 shows the frequency applied to two AODs in order to focus to astationary spot;

FIG. 43 shows how these frequencies need to be changed in order toprovide a scanning X-deflection;

FIG. 44 shows how the maximum and minimum drive frequencies in the AODslimit the scan time;

FIG. 45 shows a series of mini scans in X;

FIG. 46 shows frequency offsets being applied between mini scans;

FIG. 47 shows a geometric derivation useful in determining the frequencyoffsets;

FIG. 48 shows a four AOD system for providing a constant change of scanangle θ in the X-Y plane with time;

FIG. 49 shows the four AOD system, in which a constant change of scanangle θ is provided in the Y-Z plane.

FIG. 50 shows an embodiment of apparatus for correcting chromaticaberration;

FIGS. 51 a to 51 c show how diffractive optical elements modulate a beamwidth in accordance with the wavelength of the beam;

FIG. 52 shows the embodiment of FIG. 50 with the diffractive opticalelements axially moved;

FIG. 53 shows the spectra of laser pulse trains at three differentwavelengths;

FIG. 54 shows the effect of beam wavelength on the embodiment of FIG.50;

FIG. 55 shows an embodiment of a chromatic aberration correcting systemusing zoom lenses and diffractive optical elements;

FIG. 56 shows the embodiment of FIG. 55 and the focal length of the zoomlenses;

FIG. 57 is a graph useful for explaining the embodiment of FIG. 55;

FIG. 58 is another graph useful for explaining the embodiment of FIG.55;

FIG. 59 shows the embodiment of FIG. 55 when a wavelength of 900 nm isused;

FIG. 60 shows the embodiment of FIG. 55 when a short wavelength is used;and

FIG. 61 shows the total length of the chromatic aberration correctingsystem.

TECHNICAL BACKGROUND

In order to fully understand the invention, it is useful to explain thetechnical effects relevant to the invention. FIG. 1 illustrates theprinciple of Bragg diffraction in an acousto-optic deflector.

The acousto-optic deflector comprises a crystal 10 and a crystaltransducer 12. The crystal is preferably a high-efficiency anisotropicacousto-optic crystal such as a TeO₂ crystal. The crystal transducer 12is attached to one side of the crystal and is arranged to propagate anultrasonic acoustic wave 14 through the crystal, preferably using theslow shear mode of propagation.

An incoming laser beam 16 entering the crystal at an angle Φ₁ will bediffracted by the acoustic wave and the first order component ofdiffraction will have an angle Φ₂ as shown in FIG. 1. The first ordercomponent of diffraction is labelled 18 in FIG. 1. There will also be azeroth order component of diffraction which is simply a continuation ofthe input laser beam 16, i.e. the zeroth order of diffraction is anundeflected laser beam.

The laser beam 16 typically has a width of 10 to 15 mm and the pluralbeams illustrated in FIG. 1 are merely illustrative of a single widelaser beam.

The equation governing the angle of diffraction is:

$\begin{matrix}{{\varphi_{2} - \varphi_{1}} = \frac{\lambda_{0}f_{ac}}{V_{ac}}} & (1)\end{matrix}$

where Φ₂-Φ₁ is the angle of diffraction, λ₀ is the wavelength of thelaser beam, f_(ac) is the frequency of the acoustic wave propagating inthe crystal and V_(ac) is the velocity of the acoustic wave propagatingin the crystal. In FIG. 1, the acoustic wave has a constant frequencyf_(ac).

It is apparent from this equation that the amount of deflection that thelaser beam undergoes is directly proportional to the wavelength of thelaser beam. Thus, higher wavelength components of light will bedeflected by more than lower wavelength components.

By manipulating the acoustic wave propagating in the crystal, specialeffects can be achieved.

For example, the acoustic wave can be “chirped” such that its frequencylinearly increases or decreases with time, for example by giving it theform:

f _(ac)(t)=f _(ac)(0)+at  (2)

In this equation the constant a is known as the “chirp rate” and ismeasured in MHz per second. It is clear from this equation that thefrequency of the ultrasonic wave is a linear function of time. FIG. 2shows the situation where the chirp rate a is negative, i.e. thefrequency of the acoustic wave linearly decreases with time. As theangle of diffraction is proportional to the frequency of the acousticwave, those parts of the laser beam that are deflected by thehigh-frequency portion of the acoustic wave will be deflected more thanthose parts which are diffracted by the low frequency portion. This isillustrated in FIG. 2 and it can be seen that the effect is to focus thelaser beam at a position in the general direction of the dotted arrow 20in FIG. 2. The distance D to the focal position in the verticaldirection is given by the following equation:

$\begin{matrix}{D = \frac{V_{ac}^{2}}{\lambda_{0}a}} & (3)\end{matrix}$

As illustrated in FIG. 3, the acoustic wave moves in the direction ofarrow 24 at the acoustic wave velocity V_(ac). The focal position 22created by the converging laser beam will thus also move in thedirection of arrow 26 at the acoustic velocity. Accordingly, one AOD canbe used to focus a laser to a position that moves at the acousticvelocity V_(ac).

It is also pertinent to point out that the range of acoustic frequenciesthat may be propagated through the crystal 10 is limited because thediffraction efficiency drops rapidly outside the design range of theAOD. FIG. 4 a shows the frequency of the acoustic wave as it varies withtime and FIG. 4 b shows the frequency of the acoustic wave as it varieswith distance.

As can be see from FIG. 4 a, it is necessary to keep the frequency ofthe acoustic wave between the limits f_(min) and f_(max). It istherefore not possible to indefinitely chirp the frequency of theacoustic wave and, once the frequency reaches f_(min) it is necessary tovery quickly change the frequency to f_(max) such that the chirping cancontinue. This creates a “saw-tooth” graph in FIG. 4 a. This samesaw-tooth pattern occurs in FIG. 4 b, but it is reversed because thefrequencies present in the acoustic wave on the right-hand side of thecrystal represent frequencies at an earlier time point than thefrequencies present in the acoustic wave at the left-hand side of thecrystal.

For one design of AOD, typical values for f_(min) are 50-60 MHz andtypical values for f_(max) are 90-100 MHz. However, a special design ofAOD may be provided that is more efficient at lower frequencies, forexample 20-50 MHz, more preferably 25-45 MHz, more preferably still30-40 MHz and more preferably still 32-37 MHz. f_(min) and f_(max) maythus be chosen in accordance with these lower and upper limits. A lowrange of acoustic frequencies are useful because they minimise thedeflection provided by any one AOD and reduce the need to provide AODsthat have large acceptance angles. This allows the efficiency to be kepthigh.

For those points in time where the “fly-back” portion of the graph ispresent in the central region of FIG. 4 b or, in other words, for thosepoints in time where the discontinuity between the highest and lowestfrequency exists in the crystal of the AOD, proper focussing cannot beachieved. There are therefore certain periods of time for which the AODcannot be used for focussing. In two-photon applications, it istherefore important to measure signals induced by the laser pulses onlyat points in time where there is minimal discontinuity in chirpedfrequency across the AOD. There is therefore a “duty cycle” limitationon the AOD which duty cycle is the amount of time, expressed as apercentage, that the AOD may be used for useful focussing. It isapparent that this duty cycle will be reduced by increasing the gradientof frequency increase/decrease in FIGS. 4 a and 4 b.

The focal spot 22 can be made stationary by utilising a second AOD, asdescribed by Kaplan et al (supra) and as illustrated in FIG. 5.

In this configuration, a second AOD crystal 10 and ultrasonic transducer12 is utilised and the ultrasonic waves in the AODs are made topropagate in substantially opposite directions. In FIG. 5, the first(upstream) AOD has an ultrasonic wave propagating from the right to theleft and the second AOD has an ultrasonic wave propagating from the leftto the right. The first AOD modifies the input laser beam 16 to be afocussed laser beam 18 with the focal spot moving substantially from theright to the left and the second AOD modifies the laser beam 18 to be astationary focussed laser beam 28. As illustrated in FIG. 5, resultantfocal spot 22 does not move.

FIG. 6 shows the same set up as FIG. 5 but additionally shows theundiffracted beam (known as the “zeroth order component of diffraction”)that is transmitted through the first AOD. Due to the offset positioningof the AODs, the undiffracted beam passes well to the right of the focalspot 22 and so does not interfere with the light reaching the focal spot22. Baffles or other mechanisms may be used to cut the undiffracted beamout of the system altogether.

For the sound wave direction and diffraction order illustrated,utilising a chirp rate of zero (as shown in FIG. 1) provides a parallellaser beam. Utilising a negative chirp rate (as shown in FIG. 2)provides a converging laser beam. Utilising a positive chirp rateprovides a diverging laser beam. These three possibilities areillustrated in FIGS. 7 a, 7 b and 7 c. In any practical system the AODswill be followed by one or more lenses 70 which serve to provide furtherfocussing. Thus, whether the laser beam leaving the AOD system isconverging (FIG. 7 a), parallel (FIG. 7 b) or diverging (FIG. 7 c) thesubsequent lens system brings the laser beam to a real focus. The systemis preferably calibrated such that when the laser beam leaving the AODsystem is parallel (FIG. 7 b) the point at which the subsequent lenssystem 70 focuses the beam is designated the Z=0 point. Then, for thisconfiguration, applying a positive chirp rate moves the resultant focalpoint upwards (see FIG. 7 a) and applying a negative chirp rate movesthe focal point downwards (see FIG. 7 c). In practice, the laser beampasses through several lenses before reaching the physical target.

It will be apparent from FIG. 6 that a problem can arise when the firstand second AODs are aligned so as to be parallel. In this case, theundiffracted beam 16 can interfere with the beams reaching the focalpoint 22. This problem is alleviated in accordance with the secondaspect of the invention (please see later).

FIG. 8 illustrates how the focal spot 22 can be moved within the targetvolume. The following and subsequent explanations ignore the effect ofsubsequent lens systems (such as the lens 70 in FIG. 7) in order toprovide clarity. In any practical embodiment, such a lens system will bepresent and the principles below apply equally to the case when the AODsthemselves provide a diverging laser beam (in which case there is avirtual focus above the laser beams that is relayed by the subsequentlens optics to a negative Z position). In order to assist inunderstanding this aspect of the invention the following Figures takethe example when the chirp rate is positive which in this configurationproduces a converging laser beam.

As explained above, the distance to the focal position is inverselyproportional to the chirp rate a. Increasing the chirp rate thereforebrings the focal position upward in the Z direction and decreasing thechirp rate brings the focal position downward in the Z direction. Asexplained in FIG. 8, varying the slope of the frequency time graph (i.e.modifying the chirp rate a) serves to move the focal position 22 in theZ direction. As also illustrated in FIG. 8, the focal position 22 may bemoved in the X direction by varying the separation between the two rampsin the frequency-time graph. When the two AODs are excited with acousticwaves that are identical and without any chirp, the resultant focalposition is defined as the X=0, Z=0 position. When a chirp isintroduced, this moves the focal position in the Z direction. When theabsolute frequency of the waves applied to the two AODs differs, thiscauses the focal position 22 to be moved in the X direction.

FIG. 9 illustrates the problem of chromatic aberration and how it causesa laser beam having any sort of spectral width to be focussed to ablurred area, rather than to a distinct position in the X-Z plane.

In FIG. 9, the input laser beam 16 has a certain spectral width. Theinput laser beam might be a continuous laser beam having severalspectral components or might be a pulsed laser beam of a singlefrequency. When a laser beam is pulsed (that is to say time-windowed bymode locking the laser) this introduces a spectral width to the laserbeam. The longest wavelength component of the pulse is shown by thearrows 16, 18, 28 (displayed in grey in FIG. 9) and the shortestwavelength component of the pulse is shown by the arrows 16 a, 18 a, 28a (drawn darker in FIG. 9).

It can be seen from FIG. 9 that the longer wavelengths are diffractedthrough a larger angle.

As illustrated in FIG. 9, the focal point 22 for the long wavelengthcomponent does not coincide with the focal point 22 a for the shorterwavelength component. Wavelengths in between the two illustrated will befocussed to a point somewhere on the line linking focal spot 22 withfocal spot 22 a. The effect of the AODs is therefore to not properlyfocus a laser beam having spectral width to a unique point.

This problem can be alleviated by using longer laser pulses (which canhave a narrower spectral width). However, making the pulses longer makesthem less suitable for two-photon microscopy applications as thetwo-photon microscopy effect is predicated on being able to supply alarge number of photons in a very short space of time.

Two-Photon Microscopy System

FIG. 10 shows a two-photon microscopy system in accordance with thepresent invention.

An input laser beam 16 is passed through four acousto-optic deflectors30, 40, 50, 60 and a lens 70. The laser beams forms a focal spot 22 inthe first image field which has Cartesian axes Xi1, Yi1, Zi1. This imageis projected through other relay optics (not shown for clarity) whichcan create a second image field Xi2, Yi2, Zi2. This is projected by atube lens 80 through a microscope objective lens 90 to form a focal spot32 in the third image field Xi3, Yi3, Zi3. This third image field is thetarget field and, in two-photon applications, the target is placed inthis field. Such a target might be a slice of brain tissue or otherbiological material with a fluorescent dye that requires imaging.

The input laser beam 16 in two-photon applications takes the form of anultra-short femtosecond or picosecond pulse in order to get sufficientlyintense electric fields at the focal point. The pulses are typicallyspaced in time by a duration very much larger than the pulse length.Typical pulse lengths are 2 ps or less, preferably 500 fs or less, evenmore preferably 50 to 200 fs. The pulses are typically repeated at afrequency of 50 to 200 MHz (e.g. 80 MHz).

Two distinct experiments can be carried out with a two-photon microscopysystem. The first experiment is to image fluorescent materials and suchexperiments typically require powers of 10 mW to be focussed to an areaof just over 1 μm² (corresponding to a power density of around 600,000W/cm²). Typical laser wavelengths of 800-1000 nm (e.g. 850 nm) areutilised. The second experiment is photolysis in which the laser is usedto uncage biologically active compounds. Lasers having a wavelength of720 nm are often used and the power requirement is much higher, therebeing a need for in excess of 100 mW of power per micron squared.

In a preferred embodiment of the invention, the laser is supplied by amode locked Ti sapphire laser tuneable in the near infrared regionhaving an average power of 1 to 10 W and supplying 100 fs pulses at 80MHz.

Sensitive collection photomultipliers are utilised near to the targetarea to pick up any fluorescence from the two-photon excitation offluorophores in the target. This enables a 3D image to be constructed inimaging applications and further enables any sequence of spots in 3Dspace to be interrogated by the laser beam for repeatedly monitoring thestate of tissue at each spot during dynamic biological processes.

The AODs used in the present invention are preferably shear-modeanisotropic AODs. Suitable materials for the AOD crystal are TeO₂crystals. Such AODs rotate the polarisation of incoming laser light by90°. The AODs 30, 40, 50, 60 are schematically illustrated in FIG. 10(and in other Figures of the present application) with no interveningcomponents between them. However, in practice, such components will bepresent. Typically, these components may include half-wave plates andpolarisers (the reason will be explained later). Furthermore, atelecentric relay can be used between each AOD (as disclosed by Reddy &Saggau) to properly couple the AODs together. If such a telecentricrelay were not used, then it would be difficult to achieve a stationaryfocal position, without taking other measures.

The light emitted by the fluorophores is picked up by a photomultiplier(not shown) coupled to the system by a dichroic minor in the standardfashion.

FIGS. 11 a and 11 b graphically exemplify how chromatic aberration canbe corrected for a single point in the image field according to theprior art. FIG. 11 a shows the situation prior to correction. Thechromatic aberration has a positive magnitude for all points in theimage field and, as can clearly be seen from FIG. 11 a, the magnitude ofthe chromatic aberration varies across the image field in a generallylinear fashion. In FIG. 11 a, the chromatic aberration at the right-handside of the imaging field is larger than the chromatic aberration at theleft-hand side of the imaging field. Using the best compensation methodsknown in the art, the chromatic aberration can be corrected for a singlepoint in the image field, as shown in FIG. 11 b. The single point ishere selected to be the centre of the image field such that themagnitude of the chromatic aberration at the extremities of the imagefield is equal and opposite. This provides the least overall chromaticaberration. However, it is apparent from FIGS. 11 a and 11 b that theslope of the line defining the chromatic aberration has not at all beenchanged. The present invention discloses apparatus and methods formodifying this slope so that a chromatic aberration graph similar toFIG. 11 c can be obtained. Modifying this slope is referred to herein asat least partially correcting the magnification chromatic aberration.

Chromatic Aberration Correction

FIG. 12 shows the effect of chromatic aberration (as explained in FIG.9) for points in the first image field Xi1, Yi1, Zi1 of FIG. 10.

In this embodiment, the lens 70 has a focal length of 0.3 m and thisfocal point is allocated the zero point along the Z-axis. The zero pointalong the X-axis is the point of symmetry (i.e. the centre line) of thelens 70. The dots in FIG. 12 show positions in the image field that theshortest wavelength components of a 1 ps laser pulse at 850 nm can befocussed to by varying the chirp and frequency difference between thefirst and second AODs used to focus in the X-Z plane. The linesemanating from the dots show the points where the other frequencycomponents of the 1 ps pulse will be focussed. Thus, the end of the linefurthest from the dot represents where the longest wavelength componentsof the pulse will be focussed.

Some observations can be made about the nature of the chromaticaberration. Firstly, in common with the results of Kaplan, Salomé andReddy & Saggau (supra), there is no chromatic aberration at the pointXi1=Zi1=0. The reason for this is that, at this point, there is no net Xdeflection and the AODs are being operated with acoustic waves having asingle frequency and there is no chirp to produce Z focussing. Allfrequency components are therefore focussed to the same spot. As onemoves along the X-axis from this “compensation point” the amount ofchromatic aberration increases accordingly. Similarly, as one movesalong the Z-axis, the amount of chromatic aberration increases. Lookingat FIG. 12 as a whole, for positions in the image plane at Z<0.15 m, thechromatic aberration seems to have the effect of magnifying the longerwavelength components in the image plane more than the shorterwavelength components. In other words, if FIG. 12 were re-drawn suchthat the longer wavelength components were shown as dots, this graphwould look like a magnified version of the dots representing the shorterwavelength components. This magnifying effect of the chromaticaberration is referred to herein as magnification chromatic aberration.

FIG. 12 is illustrated for a 1 ps pulse. For even shorter pulses, suchas 100 fs, even more chromatic aberration is apparent.

Another observation from FIG. 12 is that the X dispersion (i.e. thechromatic aberration in the direction of the X-axis) reduces to zero forthe value of Z=0.15. Analysis shows that, for the case when the imaginglens 70 is very close to the final AOD, this will occur generally forvalues of Z at approximately half the focal length of the lens 70.

FIG. 13 shows a view similar to FIG. 12 although here the image is thatobtainable under an objective lens having 40× magnification. As withFIG. 12, the tails indicate the direction and relative size of chromaticaberration. In this case the system lenses have been placed intelecentric positions so that the image field is rectangular rather thantrapezoidal. As in FIG. 12, chromatic aberration has been reduced tozero for X=Z=0 but has increasing values further away from thiscompensation point.

The present invention teaches to at least partially correct themagnification chromatic aberration by utilising at least one opticalelement.

In a first embodiment, this at least one optical element can be aspecially manufactured tube lens 80 or microscope objective lens 90.

For a particular X-Y plane at a certain value of Z, all of the chromaticaberration will be in a direction that is directed radially away fromthe objective lens 90. This fact can be taken advantage of bymanufacturing the objective lens 90 so as to have a dispersive quality.That is to say, the objective lens 90 is manufactured from a materialwhich magnifies the longer wavelengths less than the shorterwavelengths. Such lenses can be made from combinations of conventionalcrown and flint glasses or from diffractive elements. If the correctamount of dispersion is introduced into the objective lens 90 (oralternatively the tube lens 80) the chromatic aberration in the whole ofthe selected X-Y plane can be substantially corrected. This can increasethe NRDV in that X-Y plane by a factor of 50 or more.

FIG. 14 shows a slice through the X-Z plane and also shows first andsecond AODs 30, 40 designed to allow focussing in this plane. Naturally,a preferred embodiment also includes third and fourth AODs for focussingin the Y-Z plane and the compensation element 80 or 90 can equallycorrect the magnification chromatic aberration in the Y direction.

The provision of a dispersive lens to correct the magnificationchromatic aberration is thus a significant advance in the art as thechromatic aberration can be corrected not just at a single point X=Y=Z=0but for a whole plane in the image field.

A more preferred embodiment, representing the best mode of operating theinvention, provides for significant correction of the magnificationchromatic aberration not just in a 2D plane but throughout the majorityof the 3D image field. This can be achieved in the present embodiment byutilising a telecentric relay to correct the magnification chromaticaberration.

The telecentric relay advantageously comprises two lenses both havingdispersive qualities. The first lens is preferably one in which thefocal length decreases with increasing wavelength. The second lens ispreferably one in which the focal length increases with increasingwavelength. Accordingly, the first lens will tend to project the longerwavelength components to a point nearer to the first lens than theshorter wavelength components. This is illustrated in FIG. 15. An image120 (here of a semi-circle with a dot at the centre of the curvature ofthe semi-circle) is projected through a dispersive lens 110 which has aquality of having a reduced focal length with increasing wavelength.Assuming that the light making up the image 120 has some spectral width,the long wavelength components will be projected to form the image 130and the short wavelength components will be projected to form the image140. As can be seen in FIG. 15, the long wavelength components areprojected to a point closer to the lens 110 than the short wavelengthcomponents 140. As a result of this, the long wavelength components 130are magnified less than the short wavelength components 140. Another wayto explain the qualities of the dispersive lens 110 is to state that ithas a negative dF/dλ wherein F is the focal length and λ is thewavelength of light being transmitted through the lens 110.

FIG. 16 shows a telecentric relay having first lens 110 and second lens150.

The second lens 150 has the quality of positive dF/dλ. In other words,the focal length for longer wavelengths is greater than the focal lengthfor shorter wavelengths. As the longer wavelength components 130 of theprojected image 120 are further away from the lens 150 than the shorterwavelength components 140, projection through the lens 150 will tend torealign the centre points of the images 130, 140 to form projectedimages 160, 170 respectively (see FIG. 16). Furthermore, because thelonger wavelength components 130 are further away from the lens 150 thanthe shorter wavelength components 140, they will be magnified less thanthe shorter wavelength components. Thus, what results is an imagecomprising long wavelength components 160 and short wavelengthcomponents 170 which are centred on one another but at which the longwavelength components are magnified less than the short wavelengthcomponents.

Such a telecentric relay can be utilised in the system of FIG. 10 toproject the first image (in the Xi1, Yi1, Zi1 coordinates) to the secondimage (not shown) or to project the second image to the third image (inthe Xi3, Yi3, Zi3 coordinates). As will be apparent from a considerationof FIG. 12, the effect of the relay in reducing the magnification of thelonger wavelength components will be to substantially correct themagnification chromatic aberration that exists in the first image.

The lenses of the telecentric relay can be made of any dispersivematerial such as combinations of conventional crown and flint glasslenses and diffractive elements. Furthermore, the invention is notlimited to utilising two lenses and more or less may be used.

FIG. 17 shows a view similar to FIG. 16 but also including themicroscope objective lens 90. As in FIG. 16, the first lens 110 has anegative dF/dλ whereas the second lens 150 has a positive dF/dλ. Theobjective lens 90 has a dF/dλ of zero. The dotted line of FIG. 17 showslight of a longer wavelength than the solid line. At the final image,the fact that the longer wavelength is focussed on the image from alarger numerical aperture shows that it has a smaller magnification.

FIG. 18 shows an alternative embodiment in which first lens 110 has anegative dF/dλ, second lens 150 has zero dF/dλ and objective lens 90 hasa negative dF/dλ. It is apparent from this diagram that, yet again, thelonger wavelengths are magnified less than the smaller wavelengths.

FIG. 19 shows a further alternative embodiment. Here, first lens 110 haszero dF/dλ, second lens 150 has a negative dF/dλ and objective lens 90has a positive dF/dλ. As in the other embodiments, the longerwavelengths are magnified less than the shorter wavelengths.

It will be apparent to one of ordinary skill in the art that variousother combinations of lenses can be utilised to achieve the technicaleffect of magnifying the longer wavelengths less. The Figures presentedherein are just some examples from a multitude of possibilities.

FIG. 20 shows the image after correction and it can immediately be seenthat the lines representing the chromatic aberration are much shorter.This translates into an increase in NRDV of over 30 times.

Using the system of the present invention, the magnification achievableis not isotropic in the X, Y and Z volume. In general the magnificationin the Z-direction is equal to the square of the magnification in the Xand Y-directions. Thus, if the X and Y coordinates are magnified by twotimes, the Z coordinates will be magnified by four times. Similarly, ifthe X and Y coordinates are magnified by 0.5, the Z components will bemagnified by 0.25.

FIG. 21 shows a graph of how the NRDV varies with a “compensationfactor” C. A compensation factor C=1 is selected to coincide with theamount of chromatic dispersion in the compensator that gives perfectcompensation for all the chromatic aberration in the Z-direction. Avalue of twice this chromatic dispersion (C=2) gives perfectcompensation in the X and Y-directions. The compensation factor can beselected in accordance with the application to which the apparatus isput. For example, if the apparatus is being applied in a 2D imagingscenario, where focussing to different points in different Z-positionsis not required, the compensation factor C can be set equal to 2 so asto achieve perfect chromatic aberration correction in the whole X-Yplane. This compensation factor also gives the highest NRDV and issuitable for imaging 3D spaces where the depth of interest remainswithin the high resolution Z range. If greater resolution imaging isrequired over the largest possible Z range then a compensation factor ofnear 1 is better albeit at the expense of some loss of resolution at theextremes of X and Y range (see FIG. 21).

The parameter C in FIG. 21 can be further defined with reference to FIG.17. In this symmetrical case, at the design mid wavelength, the rate ofchange of focal length of the first lens 110 is equal to the positiverate of change of the focal length of the second lens 150, and the inputand output beams are parallel and of equal diameter,

$C = {{\frac{{- 4}\lambda}{f_{1}}\frac{\partial f_{1}}{\partial\lambda}} = {\frac{4\lambda}{f_{2}}\frac{\partial f_{2}}{\partial\lambda}}}$

wheref₁=focal length of lens 110f₂=focal length of lens 150λ=operating wavelength

FIG. 20 shows that the longer wavelength components of the originalimage have been magnified by less than the shorter wavelength componentsof the original image. The compensation is such as to slightlyovercompensate in the Z-direction and slightly undercompensate in theX-direction (C=1.3). Depending on the application, it is possible toselect or position lenses that perfectly compensate in the X-direction(but not perfectly in the Z-direction) (C=2, see FIG. 22) or whichperfectly compensate in the Z-direction (but not perfectly in theX-direction) (C=1, see FIG. 23). The example of FIG. 20 is a compromisesolution (C=1.3).

FIGS. 24 and 25 illustrate the effect of the invention in another way.

FIG. 24 shows the NRDV for a prior art system in which the second AOD ofthe AOD pair has an acceptance angle range of ±1.5 mrad. As can be seenfrom FIG. 23, this leads to a maximum NRDV of approximately 200,000. TheNRDV is calculated as the number of distinguishable points in the imagefield where enough power can be supplied to achieve the two-photoneffect. The threshold density selected for achievement of the two-photoneffect is 600,000 W/cm² and FIG. 24 takes account of losses in eachoptical component.

FIG. 24 also shows notional graphs for laser powers of 6 W, 12 W and 24W. The best currently commercially available lasers have powers of 3 W.Thus, FIG. 24 graphically illustrates that, even if a laser having apower of 24 W was available, the target NRDV of 7.8 million could neverbe reached using the prior art systems. Indeed, FIG. 24 shows that,using prior art AOD input acceptance angles of ±1.5 mrad leads to asystem having an NRDV of approximately 200,000.

FIG. 25 shows a graph similar to FIG. 24, but taking into account themagnification chromatic aberration correction provided by the presentinvention. It is firstly apparent from FIG. 25 that, even when an inputacceptance angle range for the second AOD in the pair is selected at±1.5 mrad, the NRDV is larger than in the prior art. Furthermore, themagnification chromatic aberration correction has moved the graphs suchthat it is now possible to obtain an NRDV of 2.4 million using a 3 Wlaser. This was simply impossible in the prior art. This represents a 12times improvement in NRDV compared to the prior art. The presentinventors also believe that further optimisation can be carried out toachieve the target NRDV of 7.8 million. For example the threshold600,000 W/cm² was determined experimentally using laser pulses estimatedto be 400 fs long (to account for temporal dispersion in themicroscope). Using an optical pre-chirper (as suggested by Iyer et al)to pre-compensate the laser pulses entering the microscope would enablethe 100 fs pulses to be delivered from the objective and would thusreduce this threshold considerably and enable wider acceptance anglerange AODs to be used. This would easily enable the 7.8 million MRDVtarget to be achieved.

Zeroth Order Component Blocking

A comparison of FIGS. 6 and 8 above reveals that any zeroth ordercomponents of diffraction occurring in the first AOD of FIG. 8 will betransmitted through the second AOD and can interfere with the imagefield. The reason for this is that FIG. 8, unlike FIG. 6, has the AODsmounted in a parallel configuration such that the undiffracted beampasses in a very similar direction to the diffracted beam. This problemis alleviated by the second aspect of the invention which involves theuse of polarisers and optional half-wave plates to prevent the zerothorder components of diffraction being transmitted.

In order to be accepted and successfully diffracted by an AOD, the lightmust have the correct polarisation. In particular, for high efficiencyslow acoustic wave AODs (using for example anisotropic tellurium dioxidecrystals), the optical input polarisation needs to be aligned with thedirection of propagation of the acoustic wave. Thus, where the acousticwave is such as to cause focussing of an input laser beam in the X-Zplane, the input laser beam needs to be X polarised. Any first ordercomponents of diffraction transmitted by the AOD will have had theirpolarisation rotated by 90° such that they are Y polarised. Such lightis not compatible with the second AOD shown in FIG. 7 for example. Thus,according to this aspect of the present invention, a half-wave plate anda pair of polarisers are used, as shown in FIG. 26. Input laser beam 16having X polarisation is provided to the first AOD 30. The first ordercomponents of diffraction 18 leave the first AOD 30 in a Y polarisedstate. The undeflected zeroth order components of diffraction remain inthe X polarised state. A half-wave plate 200 is disposed after the firstAOD 30 in order to rotate the polarisation by 90°. Thus, the Y polarisedfirst order components of diffraction are now X polarised and the Xpolarised zeroth order components of diffraction are now Y polarised. AnX polariser 210 is disposed after the half-wave plate and has a functionof only allowing X polarised light to pass. Thus, the X polarised firstorder components of diffraction will pass and the zeroth ordercomponents of diffraction will be blocked (because they are Y polarisedfollowing rotation by the half-wave plate). These X polarised firstorder components of diffraction are suitable for input into the secondAOD 40 where they will by rotated by 90° to become Y polarised firstorder components of diffraction. Any undiffracted light leaving thesecond AOD 40 will be X polarised and so will be blocked by the Ypolariser 220 situated downstream of the second AOD. Thus, lightreaching the focal spot 22 will solely consist of the first ordercomponents of diffraction with any zeroth order components ofdiffraction being effectively blocked by the polarisers.

With the configuration shown in FIG. 26, the focal spot 22 is actually aline perpendicular to the page because there is no focussing in the Ydirection. If, as is preferred, focussing is also required in the Ydirection, then an identical configuration to FIG. 26 can be utilised,it being merely rotated by 90° about the Z axis. In this configuration,the first and second AODs 30, 40 perform the focussing in the X-Z planeand the third and fourth AODs 50, 60 perform the focussing in the Y-Zplane.

In this configuration, all of the AODs are mounted in parallel, that isto say the acoustic waves travelling through the AODs travel in parallelplanes (parallel to the X-Y plane). Also, in this configuration, thecomponents are mounted in the following order (in the direction of laserpropagation): First AOD, half wave plate, X polariser, second AOD, Ypolarizer, third AOD, half wave plate, Y polariser, fourth AOD, Xpolariser.

There exists an even more preferred configuration of AODs and this isshown in FIGS. 10 and 27.

FIG. 27 shows two orthogonal views of the AOD configuration. The firstAOD 30 and second AOD 40 are used to provide focussing in the X-Z plane.The third AOD 50 and fourth AOD 60 are used to provide focussing in theY-Z plane. As is apparent from FIG. 27, the AODs are configured in theorder first, third, second, fourth starting from the laser beam entryend and finishing at the laser beam exit end. This configuration ispreferred because it avoids the need to utilise half-wave plates. Notshown in FIG. 27, but preferably present in a practical embodiment, arefirst to fourth polarisers. A polariser is located subsequent to eachAOD. Laser light 16 entering the first AOD 30 will be converted into azeroth order component of X polarisation and a first order component ofY polarisation. It is desirable to only transmit the first ordercomponent. A Y polariser is therefore located after the first AOD toblock the zeroth order component. This Y polarised light is suitable forinput into the third AOD 50 in which a zeroth order component of Ypolarisation and a first order component of X polarisation is produced.A X polariser is therefore located after the third AOD 50. Such Xpolarised light is suitable for input into the second AOD 40 whichproduces a zeroth order component having X polarisation and a firstorder component having Y polarisation. A Y polariser is thereforelocated after the second AOD 40. This serves to block the zeroth ordercomponent. Such Y polarised light is suitable for acceptance by thefourth AOD 60 which produces a zeroth order component having Ypolarisation and a first order component having X polarisation. An Xpolariser is therefore located after the AOD 60 to block the Y polarisedzeroth order component. As a result, all light reaching focal spot 22 isthe result of properly diffracted first order components and noundiffracted zeroth order components can filter through the system.Furthermore, this configuration does not require a half-wave plate toadapt the polarisation at various stages.

As is well known to those skilled in the art of AODs, the precise degreeof polarisation of the first order diffracted wave, although close tolinear and at 90 degrees to the direction propagation of the acousticwave, is not exact. Particularly if the AOD crystal is cut with lessthan 2 or 3 degrees deliberate misorientation of the optic axis from thedirection of propagation of the acoustic wave, the optimised input beamand the diffracted and zero order output beams of light can be slightlyelliptically polarised so the configurations described here, which uselinear polarisers would not maximally transmit the diffracted wave norperfectly suppress the undesired undiffracted zero order components ofthe light. In such cases, to further improve performance, smallrotations of inserted half wave plates or insertion of appropriate phaseplates with small fractions of a wave correction (e.g. ¼ or 1/20 wave)may fine tune the performance of the configuration concerned. The keypoint is for the polariser after each AOD to maximally transmit thewanted diffracted first order beams and maximally suppress the unwantedzero order beam. If the polariser is before another AOD, then there maybe more polarisation state adjustment before the next AOD to optimiseits performance.

Improved Acceptance Angle Crystals

Anisotropic acousto-optic crystals utilised to manufacture AODstypically have a quoted acceptance angle for the laser light. Thecrystals themselves are optimised for maximum transmission efficiency atthis acceptance angle. For the first and third AODs in the system whichreceive laser light at a constant acceptance angle, such crystals arehighly suitable. However, a problem arises when such crystals areutilised in the second and fourth AODs because the acceptance angle willvary across a range defined by the range of deflection angles capable ofbeing carried out in the first and third AODs respectively. These knowndevices are capable of deflecting an 800 nm laser beam having 3 W ofacoustic power over ±20 mrad (17.43 mrad=1°). The efficiency oftransmission is over 80%.

FIG. 28 is a graph of the efficiency of the known AOD crystals. FIG. 29is the same graph viewed from a slightly different angle. Both of thegraphs show the diffraction efficiency for various frequencies ofacoustic wave and for various incident light angles. It can be seen fromthe graph that maximum efficiency is obtained with a centre frequency ofacoustic wave of about 95 MHz and an instant angle of about 0.121 rad.FIG. 29 shows that acceptable diffraction efficiencies can be obtainedin this crystal for a range of incident angles of approximately ±1.5mrad. If the incident angle presented to the crystals strays outsidethis range, then quite low diffraction efficiencies will be presentwhich in turn limit the energy being provided to the focal spot and thuslimits the possibility of performing the two-photon interactionsnecessary in two-photon microscopy. It has been found that thediffraction efficiency of an AOD reduces approximately in inverseproportion to its design input acceptance angle. This means that as theoverall deflection angle of the four AOD system increases from the ±3mrad (=2×±1.5 mrad) possible with the standard device pairs, theefficiency falls in proportion to the inverse square of the designeddeflection angle.

The third aspect of the invention alleviates this problem by providingan acousto-optic deflector crystal which has a reasonable diffractionefficiency across a larger range of acceptance angles. A graph similarto that shown in FIGS. 28 and 29 for the new crystal is shown in FIG.30. As can be seen, a crystal configured in this manner maintains adiffraction efficiency of at least 80% of its peak across an incidentangle range of 10 mrad. However, the peak diffraction efficiencyobtainable is not as high as with the conventional AOD. Thus, the AOD ofthe invention has a lower peak efficiency than a conventional AOD butaccepts laser beams from a wider range of angles at better transmissionefficiencies than the conventional AOD. The method by which this effectis achieved will be explained with reference to FIGS. 31 and 32. FIG. 31shows a conventional AOD crystal 10 with an ultrasound transducer 12attached to one side thereof. The ultrasound transducer 12 has a width Wparallel to the direction of light propagation of approximately 3 mm.This causes the acoustic wave 14 formed in the crystal 10 to be not verydiverging. As a result, an input laser beam 16 can be deflected tobecome laser beam 18 but only if the laser beam 16 is input within anarrow incidence angle range.

FIG. 32 shows an AOD in accordance with this aspect of the invention inwhich the ultrasound transducer 12 is made much more narrow in thedirection of light propagation. In this embodiment, the width W of theultrasound transducer 12 is 1 mm or less. As shown in FIG. 32, thiscauses the propagated ultrasound wave 14 to take on a more divergingconfiguration. This in turn means that a greater range of angles oflaser beam 16 can be accepted and successfully diffracted into laserbeams 18. Thus, the narrow crystal creates a more diverging acousticwave which allows the efficient diffraction of laser beams coming from awider range of angles than if the acoustic wave was less diverging (asin FIG. 31).

Appropriate crystal transducer widths are less than 1 mm, morepreferably less than 0.5 mm, more preferably approximately 0.25 mm orless.

Dual Transducer AODs

This aspect of the invention provides an AOD having two crystaltransducers. This is shown in FIG. 33. The first crystal transducer 12 ais configured to have a narrow width in the direction of lightpropagation and the second transducer 12 b is configured to be wider inthe direction of light propagation. In this embodiment, the transducer12 a has a width of 0.25 mm and the transducer 12 b has a width of 3 mm.An excitation source 300 is provided to supply power to the transducersand a switch 310 allows an operator to select whether then first orsecond transducer is excited.

The provision of this switch allows the AOD to be operated in one of twomodes. In the first mode, the wider transducer 12 b can be utilised andthis optimises efficiency for a narrower range of acceptance angles.This is useful in applications in which it is desirable to deliver a lotof power to a small target volume, such as uncaging (photolysis)applications. The second transducer can be selected where it isimportant to achieve reasonable transmission across a greater range ofacceptance angles, for example when a larger target volume is desired tobe imaged with a larger NRDV.

The AODs designed with two crystal transducers, as explained above, arehighly suitable for use in the second and/or fourth AODs of theinvention.

Multiple Transducer AODs

This aspect of the invention is illustrated in FIG. 34. A single AODcrystal may be provided with two or more crystal transducers. Eachcrystal transducer may be selectively utilised to help propagate theacoustic wave. In the example of FIG. 34, three crystal transducers 12a, 12 b and 12 c are shown. The width of the transducers preferablyincreases in a geometric series, for example by a factor of 2 each time.The crystal transducers preferably have the property that eachsubsequent transducer is twice as wide as its predecessor. For example,transducer 12 a can be 0.25 mm wide, transducer 12 b can be 0.5 mm wideand transducer 12 c can be 1 mm wide. By appropriate selection of theswitches 310 a, 310 b or 310 c, an effective transducer width in therange between 0.25 mm and 1.75 mm can be obtained. This allows the AODto be utilised in the manner most appropriate to the application forwhich it is used. It thus helps to provide a general purpose apparatusthat can be used for a variety of different experiments. Moretransducers can be provided if desired.

As shown in FIG. 34, when switch 310 a is in the “on” position and allother switches are in the “off” position, the driver 300 excites thecrystal 12 a only. As this crystal is quite narrow, it provides anacoustic wave W1 that has a high divergence angle. When switches 310 aand 310 b are activated, this produces acoustic wave W2 which divergesless. When switches 310 a, 310 b and 310 c are activated, this producesacoustic wave W3 which diverges still less and which has the leastamount of divergence. When the widest effective transducer is used, thisproduces an AOD with the highest efficiency but with the narrowestacceptance angle for the incoming laser beam. When the narrowesttransducer is used, this produces an AOD with a lower efficiency but abetter range of acceptable angles for the incoming laser beam.Accordingly, the width of the transducer can be selected in accordancewith the desired trade-off between the efficiency of the AOD and therange of acceptance angles. As an example, the first or third AODs in afour AOD system (i.e. the first AOD for focussing in the X-Z plane andthe first AOD for focussing in the Y-Z plane) can be provided with widetransducers to give good efficiency and a low range of acceptance angleswhereas the second AOD in each focussing pair can be provided withnarrower transducers so as to give a better range of acceptance anglesat the expense of lower efficiency.

Improved Crystal Orientation

AOD crystals are usually rotated by about 6° about the X-axis and 0°about the Y-axis. This enables the centre frequency to be increased tomaximise deflection angle range and avoids the degenerate re-diffractionof power out of the diffracted beam. Because the soundwave propagationis highly anisotropic, the 6° crystal rotation results in the soundwavepower propagating at an angle of about 50° to the Y-axis.

The crystal orientation is measured with respect to the crystal axes andthe crystal axes can be determined using an X-ray diffraction technique,as described by Young et al, “Optically Rotated Long Time Aperture TeO ₂Bragg Cell”, Advances in Optical Information Processing, IV, 1990, SPIEVol. 1296, pp 304-316.

FIGS. 32 and 33 are also representative of another aspect of theinvention in which the crystal of the acousto-optic deflector has aparticular orientation. In this orientation, the input laser beam isdefined as being the negative Z axis ([001] direction) and the crystalstructure is rotated by 2° about the X axis ([110] direction) and 3°about the Y axis ([110] direction). With this crystal orientation, thesoundwave power propagates at an angle of about 20° to the Y-axis and ithas been mathematically modelled that this reduces aberration in theimage. The 3° tilt about the Y-axis is necessary to avoid loss fromdegenerate mode.

It has also been found that reducing the centre frequency of theacoustic waves from the range of 50 to 90 MHz to the range 30 to 50 MHzimproves the diffraction efficiency with this design.

In accordance with this aspect, the crystal is oriented such thatacoustic waves propagating through it have approximately 20° betweentheir wave vector and their Poynting vector. In order to achieve properfocussing, the speed of propagation of the sound waves across the AODmust be identical whether or not the first transducer 12 a or secondtransducer 12 b is being used.

This improved crystal orientation can be utilised with the second AOD inone of the focussing pairs (i.e. the AODs labelled 40 and 60).Additionally, it may also be used with a first AOD in each of the pairs(i.e. the AODs labelled 30 and 50). It is preferable that all of theAODs in the system have this improved crystal orientation.

Any of the embodiments and aspects described herein can be provided withAODs according to this orientation.

Compact AOD Configuration

FIGS. 35 a and 35 b show a typical practical configuration for the fourAOD system shown in FIG. 27. As can be seen, each of the AODs 30, 50,40, 60 is coupled to the subsequent AOD by a telecentric relay 400. Suchtelecentric relays typically have lengths along the laser path beam of400 mm or more. As can be seen from FIG. 35 b, each telecentric relayhas a total length of 4 f, where f is the focal length of one relaylens. Typically f=100 mm. Accordingly, the requirement to utilise atleast three telecentric relays to couple the AODs together adds 1.2 m tothe total beam length of the system. As explained above, differentwavelengths of light are diffracted by different amounts. Accordingly,when the laser wavelength is changed, the AODs and telecentric relayshave to be repositioned. FIG. 35 b shows two displacements H₁ and H₂.These are the displacements of the output beam centre line compared tothe input beam centre line. This displacement varies with the wavelengthof light. With a wavelength λ=700 nm, this displacement is approximately32 mm. With a wavelength of λ=900 nm, this displacement is approximately40 mm. Consequently, when changing the laser wavelength from 700 nm to900 nm, the optical components have to be realigned by 8 mm. Suchrealignment is a necessary consequence of utilising telecentric relays.Accordingly, telecentric relays are not ideal in a system for which itis intended to change the laser wavelength frequently. This aspect ofthe invention thus provides a means for dispensing with the telecentricrelays and thus allows a more compact and configurable system to beprovided.

The telecentric relays provided in the prior art are necessary to coupletogether the AODs appropriately. As shown in FIG. 8, the first AODmodulates the input laser beam 16 to be a laser beam 18 having a curvedwavefront. This wavefront is moving at the speed of sound in the Xdirection. The second AOD modulates the incoming laser beam 18 to be alaser beam 28 with a curved wavefront. The curvature here will be equalto the sum of the curvature brought about by the first AOD added to thecurvature brought about by the second AOD. The resulting focal position22 will only be stationary if the curvature endowed on the laser beam bythe second AOD equals that of the wavefront as it enters the second AOD.In the absence of the second AOD, it is apparent from FIG. 8 that thecurvature of the laser beam 18 increases as you move further away fromthe first AOD. When the AODs are set up to endow an incoming laser beamwith the same curvature (i.e. the AODs are set up with the same ramprates), it is thus necessary to either place the AODs extremely closetogether or to telecentrically relay the output of one AOD to the inputof the next AOD.

This aspect of the present invention is based on the realisation thatthe AODs 30, 40 can be excited with different acoustic waves so as toallow realistic practical separations between the AODs without therequirement of a telecentric relay. The acoustic waves can be modifiedeither to allow the generation of a completely stationary focal position22 or precisely controlled scanning.

In FIG. 36, d₁ is the separation between the first AOD 30 and the secondAOD 40 and d′₂ is the distance from the second AOD to the focal point22.

This aspect of the invention is based on the appreciation that thecurvature of the wavefront arriving at the second AOD 40 must exactlymatch the additional curvature induced by the second AOD 40. As isapparent from FIG. 36, as the distance d₁ increases, the curvature ofthe arriving wavefront increases because the light is convergingdownwards towards a focus. This is compensated for in the presentinvention by providing a less rapid ramp (chirp) on the first AOD 30than on the second AOD 40. This is illustrated in FIG. 37 where it canbe seen that the ramp rate a₁ for the first AOD 30 is lower than theramp rate a₂ for the second AOD 40 (a₁ is equal to the gradient of theline 31 and a₂ is equal to the gradient of the line 41). This serves toproduce a focal position 22 which is stationary in the X direction, asshown in FIG. 37.

Referring to FIG. 38, the first AOD 30 is excited with an acoustic wavehaving a chirp rate of a₁. Accordingly, an incoming laser beam 16 isconverted to converging laser beam 18 that is focussed at the point 23 adistance d′₁ from the first AOD 30. As is well known, this distance d′₁is given by:

$\begin{matrix}{d_{1}^{\prime} = \frac{V^{2}}{\lambda \; a_{1}}} & (4)\end{matrix}$

wherein:V=speed of sound in the AODs (m/s)a₁=ramp rate of first AOD drive (Hz/s)k=wavelength of light (m)

It follows from this that the radius of the curvature of the wavefrontof the laser beam 18 at the point where it meets the second AOD 40 isgiven by:

d′ _(1−d) ₁  (5)

In order that the resulting focal position 22 is stationary, thecurvature added to the laser beam 18 by the second AOD 40 must equal thecurvature of the laser beam 18 as it arrives at the second AOD 40.Accordingly:

$\begin{matrix}{d_{2}^{\prime} = \frac{d_{1}^{\prime} - d_{1}}{2}} & (6)\end{matrix}$

The factor of 2 appears in this equation because the curvature added bythe second AOD 40 is identical to the curvature that already exists atthe laser beam 18 as it enters the second AOD 40. The resultingcurvature of the laser beam 28 is thus twice the curvature of the laserbeam 18. From these equations, it can be deduced that:

$\begin{matrix}{a_{2} = \frac{V^{2}}{2\lambda \; d_{2}^{\prime}}} & (7) \\{a_{1} = \frac{V^{2}}{\lambda ( {{2d_{2}^{\prime}} + d_{1}} )}} & (8) \\{\frac{a_{1}}{a_{2}} = \frac{2d_{2}^{\prime}}{{2d_{2}^{\prime}} + d_{1}}} & (9)\end{matrix}$

In these equations, d₁ is always a positive value. The values d′₂, a₁and a₂ are positive for converging rays as shown in FIG. 7 a andnegative for diverging rays as shown in FIG. 7 c. As explained earlier,even when the rays are diverging, a real focal position is achievedusing subsequent optics, such as the lens 70.

When equation (9) is studied, it is apparent that if d₁ is made to bezero then a₁ equals a₂. This is the assumption utilised in the prior artbecause coupling two AODs together with a telecentric relay exactlycouples the output of the first AOD onto the input of the second AOD andthus gives an effective separation of the AODs of zero. Up until now, ithas always been thought that the frequency chirp across the two AODsought to be the same and that the effective separation between the AODsshould be zero (by virtue of utilising a telecentric relay). Theequations derived by the present inventors show that the chirp rateacross the two AODs can be made slightly different, in accordance withequation (9), to account for a real separation of d₁ between the twoAODs, to provide a system which provides a stationary focal position 22without a telecentric relay between the AODs.

This is achieved by adjusting the ramp rate a₁ of the first AOD 30, inaccordance with equation (8), to allow for the change in wavefrontcurvature between the first AOD 30 and the second AOD 40. Preferably,the wavefront curvature arriving at the second AOD 40 equals theadditional curvature that is added by the second AOD 40. This “matchingof curvature” provides for a stationary focal position.

In the equations and analysis above, the distances are apparent opticalthicknesses. If further optical components are interposed between theAODs, such as half wave plates and polarisers, then the apparent opticalseparation needs to be calculated by taking into account the refractiveindex of such additional components. Also, the refractive index of theAODs themselves needs to be taken into account. This can be done byassuming that the acoustic wave enters and leaves the AOD at itsthickness-midpoint such that the apparent optical distance d₁ is equalto the distance in air between the AODs plus half the thickness of thefirst AOD 30 divided by its refractive index plus half the thickness ofthe second AOD 40 divided by its refractive index. When the two AODs areidentical, then the value d₁ equals the distance in air plus thethickness of the AOD divided by its refractive index.

These principles can be extended to a system which utilises four AODs tofocus in more dimensions. As discussed above, when two AODs are used, asshown in FIG. 38, the focal position 22 is a line extendingperpendicularly out of the page. Four AODs can be utilised to focus inboth X and Y to produce a point focal position 22.

FIGS. 39 a and 39 b show two orthogonal views of a preferred four AODsystem. As in FIG. 38, the first AOD 30 is separated from the second AOD40 by a distance d₁ and the second AOD 40 is a distance d′₂ from thefocal point 22. In addition, third and fourth AODs 50, 60 are provided,the distance between the third AOD 50 and the fourth AOD 60 being d₃ andthe distance from the fourth AOD 60 to the focal point 22 being d′₄. Theramp rates for the third and fourth AODs can be calculated in a similarway as for the first and second AODs. Very similar equations apply:—

$\begin{matrix}{a_{4} = \frac{V^{2}}{2\lambda \; d_{4}^{\prime}}} & (10) \\{a_{3} = \frac{V^{2}}{\lambda ( {{2d_{4}^{\prime}} + d_{3}} )}} & (11) \\{\frac{a_{3}}{a_{4}} = \frac{2d_{4}^{\prime}}{{2d_{4}^{\prime}} + d_{3}}} & (12)\end{matrix}$

Accordingly, in the four AOD system, the first and second AODs arestimulated in the same way as the first and second AODs of the two-AODembodiment. This provides the necessary focussing in the X-Z plane. Inaddition, third and fourth AODs are stimulated such that the curvatureof the wavefront arriving at the fourth AOD equals the additionalcurvature added by the fourth AOD, hence doubling the curvature of thewavefront as it leaves the fourth AOD. This provides the necessaryfocussing in the Y-Z plane. The distances d′₂ and d′₄ are selected toensure that the final focal spot position 22 is as desired. As will beapparent from FIGS. 39 a and 39 b, the actual distances between the AODsand the optical thickness of any intervening components, as well as theAODs themselves, needs to be taken into account when determining d₁, d₃,d′₂ and d′₄.

Depending on the exact configuration used, further fine tuning may beapplied to achieve an exactly stationary spot. The equations above arebased on the simplified assumption of AOD crystals having surfaces thatare approximately perpendicular to the direction of propagation of thelight. It is possible to manufacture the AODs with slightly angled faces(and there are practical reasons to do exactly this) and this can causeerrors in the separations used in the equations that can result in asmall residual movement of the focal position. These residual movementscan be corrected by small adjustments to the ratio of ramp rates a₁/a₂,a₂/a₄. These corrections can either be found experimentally or bybuilding an accurate optical model using a commercial programme likeZemax. When such angled faces are used, typical corrections are muchless than +/−2% to the ramp rate of each AOD. Similarly, smallcorrections may be applied to the ratio of the X ramp rate to the Y ramprate to fine tune the astigmatism of the focal position 22. This isequivalent to adjusting the ratio of d′₂ to d′₄ so that the Z value ofthe focal position in the X-Z and X-Y planes is the same. These finetuning corrections are a function of the Z position of the focal spotand can readily be built into the algorithms that compute the ramp rateof the AODs before each scan.

As will be understood from the above, it is possible with the presentinvention to utilise two or four AODs to achieve a completely stationaryfocal line or focal point inside or on a target. This can be achievedwithout lengthy telecentric relays between the AODs by appropriatemanipulation of the ramp rates of the acoustic waves applied to theAODs. The resulting system can thus be used to achieve random accessfocussing at very fast speeds. For example, it is possible torepetitively focus to 30 different positions within or on the target ata frequency of 1000 Hz. In other words, in one second, the laser beamcan be focussed to 30 points one thousand times. To achieve this, thelaser beam focal point is repositioned 30,000 times in one second. Thisis simply not achievable with prior art galvanometer minors.

Scanning a Target

To build up a three-dimensional image of a target, it is useful to beable to follow a raster scan with the focal point along a predeterminedpath through the target. One potential raster scan is to move the focalpoint in the X direction, keeping the Y and Z values constant, to thenincrement the Y position by some small amount, to perform another scanin the X direction and so on until a two-dimensional grid of scans isachieved. Thereafter, the Z direction is incremented and anothertwo-dimensional grid is scanned until a three dimensional volume hasbeen built up. This can be done quite quickly with the system of thepresent invention such that a three-dimensional image can be provided.

One problem encountered when implementing a raster scan using the systemof the present invention is that there are minimum and maximum limits onthe frequency of acoustic waves that can be put through the AODs. Thisis illustrated as f_(max) and f_(min) in FIG. 4 a, for example. Typicalvalues are 30 Mhz for the minimum frequency and 40 Mhz for the maximumfrequency. As shown in FIG. 4 a, there will be a “flyback” portionwhereby the frequency is suddenly switched from the maximum frequency toa lower frequency (in the case of applying a positive chirp rate) or asudden switch from the minimum frequency to a higher frequency (in thecase when applying a negative chirp rate). The X, Y and Z positionsdepend on the chirp rate and the difference in absolute frequenciesbetween the first and second AODs (see FIG. 8). Accordingly, this suddenchanging of the absolute values of both frequencies will not cause amovement in the X Y and Z position of the focal spot if it isimplemented properly.

If desired, the X and Y scans can be carried out simultaneously with theY scan being much slower than the X scan. This leads to thetwo-dimensional scanning pattern shown in FIG. 40.

FIG. 41 shows how a raster scan performed in the X-Y plane can beachieved.

Movements in X or Y correspond to changes in the angle of the outputlaser beam from the AOD system.

As is apparent from the above equations and from FIG. 42, to focus astationary spot at X=0 requires a₂ to be slightly larger than a₁ inaccordance with equation (9). The situation shown in FIGS. 41 and 42 iswhere the focal position has a positive value of Z such that the laserbeam converges upon exiting the AOD system. In order to provide an Xdeflection that moves at some constant linear velocity, it becomesnecessary to vary a₁ and a₂ such that there is a linearly increasingdifference between the absolute frequency of the acoustic wave in thefirst AOD and the absolute value of the frequency in the second AOD.This is illustrated in FIG. 43 where the dotted lines show a₁ beingreduced and a₂ being increased. This provides a scanning X deflectionwhereby the X value of the focal point decreases linearly with time asshown in the lower part of FIG. 43.

As shown in FIG. 8, varying the slope a₁, a₂ of the ramps varies the Zposition. For Z=0, a₁ and a₂=0 and X scanning can easily be achieved bymaking a₁ slightly negative and a₂ slightly positive. For other valuesof Z, higher magnitudes of a₁ and a₂ are required and the limits on theminimum and maximum drive frequencies of the AODs mean that it ispossible to hit one of the limits very quickly. For non-zero values ofZ, it is possible to hit either the minimum or maximum drive frequencybefore one has completed an X scan across the target. In such cases, itis convenient to perform a series of X “mini-scans” in which the X scanas a whole is interrupted at various points in time to allow thefrequency to be reset in a “flyback” period. This is illustrated in FIG.44, which is a cropped version of FIG. 42 showing how the maximum andminimum drive frequencies limit the amount of X deflection that can takeplace.

FIG. 45 illustrates a series of mini-scans in X. The frequency of theacoustic wave in the first AOD 30 is designated by line 31 and thefrequency of the acoustic wave in the second AOD 40 is designated by theline 41. In this example, line 31 has a shallower gradient than line 41which means that a₁ is less than a₂, which from equation (9) means thatd′₂ is positive, which in turn means that this situation is for somepositive value of Z. As shown in FIG. 45, when the absolute value of thefrequency of the acoustic wave in the first AOD 30 reaches the maximumvalue f_(max) it becomes necessary to reset the frequencies. As isapparent from FIG. 45, it will not be possible to change the frequencyin the first AOD 30 to be f_(min) because the frequency in the secondAOD 40 must be less than the frequency in the first AOD 30 to ensurethat the difference in frequencies between the two AODs continues togive the correct value of X. Accordingly, the frequency in the secondAOD 40 is reduced to f_(min) and the frequency in the first AOD 30 isreduced by the same amount. This frequency resetting takes place in aperiod of non-active time when the laser is switched off (or at leastwhen measurements are not recorded or are ignored). The non-active timegenerally has two components. The first component, known as the “resettime” is the time it takes to reset the frequency from the maximum valueto the new value or from the minimum value to the new value. This istypically 4 μs. The second component, known as the “AOD fill time” isthe time it takes to fill the AOD with appropriate acoustic waves. Thisis typically equal to the width of the AOD divided by the speed of theacoustic wave in the AOD. For example, if the AOD is 15 mm wide and theacoustic waves travel at 600 m/s, then the AOD fill time will be 25microseconds. The total non-active time is thus typically around 30 μs.

After the AOD fill time has elapsed, the frequencies in the AODs shouldbe different by an amount equal to the difference at the end of theprevious mini-scan. As the frequencies are different by the same amount,it might be expected that the X position would be the same at thebeginning of the second mini-scan as it was at the end of the firstmini-scan. This is true when the AODs are telecentrically relayed.However, it has been found that this is not the case when there is someseparation between the AODs. Instead, the X position is different tothat which is expected as shown in FIG. 45, bottom graph. There it canbe seen that the X position is different at the start of the secondmini-scan than at the end of the first mini-scan.

The present inventors have found that the reason for this lies in theassumption that the separation of the ramps alone causes the variationin X. This assumption is only true if there is no physical separationbetween the AODs, or alternatively, if the AODs are coupled bytelecentric relays. In the case where there is an actual separationbetween the AODs, a more complicated algorithm needs to be utilised tocalculate the frequency offsets necessary to maintain the position in Xbetween the end of one mini-scan and the start of the next mini-scan. InFIG. 45, the frequency offsets are calculated by making the frequency inthe second AOD zero and reducing the frequency in the first AOD by someamount that causes the frequency difference to be the same at the startof the next mini-scan. In fact, different offsets will be needed foreach AOD as illustrated in FIG. 46. Here, the frequency in the first AODis reduced by Δf₁ and the frequency in the second AOD is reduced by Δf₂.This second frequency reduction, Δf₂ is calculated as the offset neededto reduce the frequency in the second AOD to f_(min). It has been foundthat Δf₁ and Δf₂ are related by the following equation:

$\begin{matrix}{\frac{\Delta \; f_{2}}{\Delta \; f_{1}} \approx \frac{d_{1}^{\prime}}{d_{1}^{\prime} - d_{1}}} & (13)\end{matrix}$

This equation can be proved by referring to FIG. 47. The centre line ofthe first AOD is referenced 30 and the centre line of the second AOD isreferenced 40. The ultimate focal point is shown at 22. If a frequencyoffset is introduced into the acoustic wave of the first AOD this willproduce an angular deflection of Δθ₁ at the central position (X=0) ofthe AOD. This is graphically illustrated in FIG. 47 by the angle Δθ₁between the line of the laser beam before deflection (verticallydownward in FIG. 47) and the line 35 of the laser beam after deflection.However, at the second AOD, at the position X=0 the ray will apparentlybe deflected by a different angle Δθ₂. As is apparent from FIG. 47, Δθ₂is larger than Δθ₁. A geometric deduction leads to the equation (whichis valid for all small angles of Δθ₁ and Δθ₂):

$\begin{matrix}{\frac{{\Delta\theta}_{2}}{{\Delta\theta}_{1}} \approx \frac{d_{1}^{\prime}}{d_{1}^{\prime} - d_{1}}} & (14)\end{matrix}$

Equation (14) is derived once it is realised that the change in anglesof the beam is directly proportional to the change in frequency. Asshown in the lower part of FIG. 46, applying offsets that have therelationship of equation (13) means that the X deflection is not changedbetween the end of one mini-scan and the beginning of the nextmini-scan. Please note that FIG. 46 does not show any AOD fill time forclarity although there will in reality be an AOD fill time as shown inFIG. 45. In practice, Δf₁ and Δf₂ are calculated to be correct at theend of the AOD fill time, when the data collection restarts. Convertingequation (13) to the nomenclature of FIGS. 39 a and 39 b, we find:

$\begin{matrix}{\frac{\Delta \; f_{1}}{\Delta \; f_{2}} = \frac{2d_{2}^{\prime}}{{2d_{2}^{\prime}} + d_{1}}} & (15)\end{matrix}$

Similarly, when there are four AODs present:

$\begin{matrix}{\frac{\Delta \; f_{3}}{\Delta \; f_{4}} = \frac{2d_{4}^{\prime}}{{2d_{4}^{\prime}} + d_{3}}} & (16)\end{matrix}$

The scan rate δθ/δt can be calculated as follows:

For the simple case where the AODs are coupled by telecentric relays(i.e. d₁ is considered to be zero) the scan rate is proportional to thedifference in slopes of the chirp signal provided to each AOD. In fact,the scan rate is given by:

$\begin{matrix}{\frac{\delta\theta}{\delta \; t} = {\frac{\lambda}{V}( {a_{1} - a_{2}} )}} & (17)\end{matrix}$

In the simple case of d₁=0, the following equations result for the ramprates a₂ and a₁.

$\begin{matrix}{a_{2} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}} & (18) \\{a_{1} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} - {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}} & (19)\end{matrix}$

It can be seen from these equations that the ramp rate a₂ is increasedby the same amount that the ramp rate a₁ is decreased. This, however,only applies when d₁ is considered to be zero (i.e. when the AODs aretelecentrically coupled). In the more complicated case when d₁ isnon-zero. The values for a₁ and a₂ are instead given by:

$\begin{matrix}{a_{1} = \frac{\frac{V}{\lambda}( {\frac{V}{d_{2}^{\prime}} - \frac{\delta\theta}{\delta \; t}} )}{( {2 + \frac{d_{1}}{d_{2}^{\prime}} - {\frac{d_{1}}{V}\frac{\delta\theta}{\delta \; t}}} )}} & (20) \\{a_{2} = {\frac{V^{2}}{2\lambda \; d_{2}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}} & (21)\end{matrix}$

These equations apply where there are two AODs for focussing in the X-Zplane or, as shown in FIG. 48, when there are four AODs. In this case,the angular scan rate δθ/δt is that measured about the second AOD 40.The apparent rate as measured about the last AOD 60 can be obtained ymultiplying this scan rate by d′₂/d′₄.

FIG. 49 shows the appropriate equations for the Y-Z plane. Here, Φ isthe angle as measured from the fourth AOD 60.

$\begin{matrix}{a_{3} = \frac{\frac{V}{\lambda}( {\frac{V}{d_{4}^{\prime}} - \frac{\delta\varphi}{\delta \; t}} )}{( {2 + \frac{d_{3}}{d_{4}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}} )}} & (22) \\{a_{4} = {\frac{V^{2}}{2\lambda \; d_{4}^{\prime}} + {\frac{V}{2\lambda}\frac{\delta\theta}{\delta \; t}}}} & (23)\end{matrix}$

As will be appreciated from FIGS. 39 a and 39 b, the spacing betweeneach adjacent AOD is related to the distances d₁, d₃, d₂′ and d₄′ by thefollowing equations:

The effective separation between the first AOD 30 and the third AOD 50is:

d ₁ +d′ ₂ −d ₃ −d′ ₄

The effective separation between the third AOD 50 and the second AOD 40is:

d ₃ +d′ ₄ −d′ ₂

The effective separation between the second AOD 40 and the fourth AOD 60is:

d′ ₂ −d′ ₄

Multi-Wavelength System

FIGS. 20 to 23 above show the possibility of varying the compensationfactor C to either fully compensate for chromatic aberration in theZ-plane (FIG. 23), to fully compensate for chromatic aberration in the Zand Y planes (FIG. 22) or to achieve some intermediate compensation inwhich chromatic aberration is compensated in all planes, but not for themaximum extent (FIG. 20). In order to vary this compensation factor C,it is necessary to vary the strength of the lenses used in thetelecentric relay. For example, the lenses 110 and 150 in FIG. 17 can bereplaced with more strongly or less strongly dispersive lenses in orderto vary the compensation factor C. In any practical embodiment of asystem, it would be beneficial to design into the system a method forvarying the compensation factor C, which does not involve having tophysically replace lenses.

Another problem lies in the fact that it is desirable for mostneuroscience applications to be able to select the wavelength ofelectromagnetic radiation that is used. Typically, wavelengths of 690 to1000 nm are used in neuroscience applications. Changing the wavelengthof a laser beam passing through diffractive optics automatically changesthe deflection angles introduced by such diffractive optics (because thedeflection angle is proportional to the wavelength). Accordingly, whileit is straightforward to design a system that can operate at a singlelaser wavelength, it is more difficult to design a system that canoperate across a range of wavelengths.

One possible approach is to design the system to operate at the maximumwavelength (e.g. 1000 nm). In such a system, the maximum diffractionangles would be catered for and it can be ensured that no light is lostby light being diffracted through a greater angle than designed. Theserious drawback of such a system is that the system inherently worksnon-optimally for any wavelength less than 1000 nm. In particular, dueto the smaller deflection angles, the aperture of the objective lenswill not be filled when wavelengths of less than 1000 nm are used andthis seriously reduces the amount of power that can be introduced to thetarget at the focal position.

It would be desirable to design a system in which the electromagneticradiation wavelength can be varied without influencing the amount ofpower delivered to the target or the intensity of the focus provided.

FIG. 50 shows one embodiment of the system shown in FIG. 17. Here, thefirst optical element 110 is provided by a lens 112 having a positivefocal power and a compensation plate 111. The second optical element 150is provided by a lens 152 having a positive focal power and a secondcompensation plate 151. Compensation plate 111 is comprised of apositive focal length diffractive optical element 115 intimatelyattached (e.g. glued) onto the plane surface of a negative focal lengthconventional lens. Compensation plate 151 is comprised of a negativefocal length diffractive optical element 155 intimately attached (e.g.glued) onto the plane surface of a positive focal length conventionallens 156. Accordingly, the combined effect of compensation plate 111 andlens 112 is to have dF/dλ negative and the combined effect ofcompensation plate 151 and lens 152 is to have dF/dλ positive.

The lenses 112 and 152 have approximately the same effect on allwavelengths of light. Accordingly, virtually all of the chromaticaberration correction is achieved by the compensation plates 111 and151. FIGS. 51 a to 51 c show the effect of changing the laser wavelengthon the chromatic aberration correction of the compensation plates 111and 151.

At the design centre wavelength, as shown in FIG. 51 a, eachcompensation plate 111 and 151 provides no overall lensing effect. Thisis because at this wavelength, the focal length of the diffractiveoptical elements 115, 155 is exactly equal and opposite to the focallength of the attached lens 116, 156. Accordingly, light at the designwavelength passes through the compensation plates like a flat glassplate.

As the wavelength of laser light is increased from the designwavelength, the diffractive optical elements 115, 155 become stronger(the diffraction angle is proportional to the wavelength) so as toproduce a lensing effect as shown in FIG. 51 b.

At wavelengths less than the design wavelength, the diffractive opticalelements become weaker, producing the lensing effect shown in FIG. 51 c.It is apparent from FIGS. 51 a-51 c that the diameter of the outputlaser beam will be influenced by the centre wavelength of the laser, dueto the varying lensing effect of the diffractive optical elements 115,155.

One advantage of the arrangement shown in FIG. 50 is that the positionsof the compensation plates 111, 151 can be altered to alter the degreeof chromatic aberration correction. This applies only at the designwavelength because moving the diffractive optical elements closertogether or further apart at the design wavelength does not introduceany additional lensing (the diffractive optical elements act like a flatglass plate, as shown in FIG. 51 a at the design wavelength).Accordingly, the degree of chromatic aberration correction, C, can bevaried by simply moving the axial positions of the compensation plates111, 151 at the design wavelength. There is thus no need to replace anylenses when altering the compensation factor C. In FIG. 52, a value of Cless than 1 is shown. In FIG. 50, a value of C=2 is shown. Accordingly,slowly moving the correction plates 111, 151 from the position shown inFIG. 50 to the position shown in FIG. 52 will slowly vary the degree ofchromatic aberration correction from a correction of C=2 (which willprovide the correction shown in FIG. 23) to a value of C=1 (which willprovide the correction shown in FIG. 22) or less.

As explained above with regard to FIGS. 51 a-51 c, the compensationplates 111, 151 can be moved to independently adjust the chromaticaberration correction, C, only at the design wavelength. At otherwavelengths, the plates will introduce some amount of lensing and thusmoving them will change the output beam diameter. FIG. 53 shows atypical range of wavelengths over which it is desirable to operate asystem. It shows three potential laser spectra, a first spectrum 81having a width of 10 nm centred on 850 nm, a second spectrum 82 having awidth of 10 nm centred around 980 nm and a third spectrum 83, alsohaving a width of 10 nm centred around 720 nm. These laser pulse streamshaving spectral widths of 10 nm are typical for pulses having 100 fsduration in the time domain.

The problem is therefore how to design a system which providessufficient differential magnification to correct the chromaticaberration due to the spectral width of a single laser pulse stream butwhich yields a constant output beam size whatever the centre wavelengthof the laser pulse stream.

FIG. 54 illustrates the problem diagrammatically. At the 850 nm designwavelength, the objective lens 90 is more or less fully filled. Thismeans that the spot 22 will be of minimal size (or, in other words, thespot will be maximally focussed). At longer wavelengths (e.g. 1000 nm)the chromatic aberration correction mechanism causes the laser beams tobe deflected more and some light leaving the compensation plate 151 isnot captured by the subsequent optics. As light is lost, the powerprovided to the focal position 22 will be reduced. At shorterwavelengths, e.g. 700 nm as shown in FIG. 54, the laser beams will bedeflected through a smaller angle and the focal position 22 will becreated from a smaller diameter beam at the objective lens 90. As theobjective lens is under-filled, the diameter of the diffraction limitedfocal spot 22 will be increased compared to the minimum it could be atthis wavelength and the intensity of the spot will therefore be lessthan it could be.

For neuroscience applications, one important use of 2-photon microscopesis for uncaging neurotransmitter chemicals to selectively exciteparticular neurons. This generally requires much higher light intensityat the focal position than 2-photon imaging. It has been found that themost effective molecules for 2-photon uncaging are sensitive around theshort wavelength end of a Ti-sapphire operating range (around 600-750nm). It is therefore highly desirable to be able to fill the aperture ofthe objective lens at short wavelengths.

If the compensator is designed to fill the aperture at 1000 nm (suchthat no light is lost at this wavelength, see FIG. 54) the diameter ofthe beam at 700 nm will be only about 70% of that at 1000 nm. This wouldproduce a focal spot of 1.4 times the diameter which corresponds to afocal spot having twice the area of that produced by a properly filledobjective lens. The light at the focal position would therefore havehalf the intensity and one quarter of the 2-photon excitation ratecompared to the situation where the aperture is filled. It is thereforedesirable to fill the aperture at the lower end of the wavelength rangeand at the same time not lose any light at the upper end of thewavelength range. It would be preferable to design a system in whichthis can be achieved while at the same time allowing the degree ofchromatic aberration compensation to be adjusted at least over the rangeC=1-2 and which does not require any lenses to be removed or replacedduring use. Preferably, lenses would be simply moved within the systemto provide the correct configuration.

One solution for achieving this is shown in FIG. 55. In this system, thelens 112 in FIG. 54 is replaced by a pair of zoom lenses 113, 114.Similarly, the lens 152 in FIG. 54 is replaced by a pair of zoom lenses153, 154. The system of FIG. 55 thus includes a first pair of positivefocal length zoom lenses 113, 114, a pair of compensation plates 111,151 and a second pair of positive focal length zoom lenses 153, 154. Thepositions of the zoom lenses 113, 114, 153, 154 are adjustable to altertheir effective focal length. Further, the separation between the twocompensation plates 111, 151 is adjustable to vary the compensationfactor C and also to take account of different wavelengths of lightbeing utilised in the device. FIG. 55 shows the nomenclature used in thefollowing description to describe the lenses L and the diffractiveoptical elements D.

A method for designing a zoom compensator will now be described.Firstly, the aperture diameter d3 of the diffractive optical elements115, 155 can be selected to be a fraction R of the diameter of the inputbeam d1. It has been found that a fraction R=0.5 allows a system to bedesigned that covers a 700-900 nm range of wavelengths. A smallerfraction can be selected to cover a wider proportionate range.

The maximum separation of s3 of the compensation plates 111, 151 canthen be selected to be greater than the minimum working f number (theratio of focal length to diameter of a lens) of the diffractive opticalelement (and other lenses) multiplied by the diameter d3.

For a maximum compensation factor (C=2) the focal length of the lensesL3 and L4 can be made equal to the maximum separation s3 of thecompensation plates. As described above, L3 has negative focussing powerand L4 has positive focussing power, and the focal lengths of both L3and L4 are equal to s3.

The focal length of the diffractive optical elements (F(D3) and F(D4))at the mid-point wavelength can be made equal to the maximum separations3. F(D3) will be positive and F(D4) will be negative. This ensures thatat the mid-point wavelength, the compensator plates have zero lensingpower.

In use, the zoom lens spacings s1 and s2 are adjusted for each operatingwavelength so that parallel input light is focussed to the mid-pointbetween L3 and L4. The separations s4 and s5 are then adjusted to givethe required constant parallel output beam diameter. The spacings s1 ands2 can be calculated by calculating the effective focal lens of the zoomlens needed to transform between the correct diameter parallel externalbeams and the central focal point, given the strength of the compensatorplates at the wavelength concerned.

For example, at the mid-point wavelength where the compensator plateshave zero power, if R=0.5 then the effective focal length of the inputand output zoom lens pairs is equal to the separation s3. This followssimply because the distance from the centre point to the compensator iss3/2 and as R=0.5 the light cone diameter is doubled before becomingparallel. This is shown in FIG. 56.

The effective focal length of the zoom lenses can be calculated fordifferent operating wavelengths by taking into account the extrarequired divergence or convergence of the light cone outside of thecompensating plates. At a shorter operating wavelength (e.g. 700 nm), s3needs to be made longer because the beam will not be diffracted throughsuch a large angle with shorter wavelengths. Similarly, for longerwavelengths, the distance s3 needs to be made shorter because the beamswill be diffracted more at longer wavelengths. Once s3 has beenselected, it is possible to calculate the focal length of the first zoompair, FT1 and the focal length of the second zoom pair, FT2. In general,a longer s3 will require a longer FT2 and a smaller s3 will require asmaller FT2. The positions of the zoom lenses to meet this condition canthen be determined using standard textbook geometric optic equations forzoom lenses.

FIGS. 57 and 58 show actual values for an example when the chosenseparation s3 is 75 mm at a centre wavelength of 800 nm. It can be seenthat, at the centre wavelength, FT1=FT2=s3. For longer wavelengths, allof these values are reduced and for shorter wavelengths all of thesevalues are increased.

FIG. 58 shows how the values of s2 or s4 and the values of FT1 and FT2vary as s1 or s5 vary utilising two 125 mm focal length lenses.Accordingly, it is possible to set FT1 and FT2 and thereafter derive s1,s2, s4 and s5.

FIG. 59 shows the positions of the lenses for a longer laser wavelengthof 900 nm. FIG. 60 shows the lens positions of a wavelength for ashorter wavelength of 700. It will be observed by comparing FIGS. 59 and60 that the diameter of the output beam is substantially the samewhether a long or short wavelength is used. Furthermore, due to thepositioning of the compensation plates 111, 151, the same amount ofselectable chromatic aberration correction C is achieved in both cases.

It will be appreciated that the zoom compensator of the presentinvention enables an aperture within the system (for example theobjective lens aperture) to be fully filled even when the centrewavelength of the laser beam is changed. In general, the compensator canbe modified to fill any aperture in the system. The system aperture isdefined here as the diameter of the maximum diameter optical beam at theentrance of the system (or subsystem concerned) that can get through thecomplete system to the final image without any of the rays being cut offby any intermediate apertures internal to the optical system.Accordingly, the corrective optics of this zoom compensator is capableof ensuring that the beam fills the same design system aperture forsubstantially all wavelengths falling within the wavelength range ofinterest. This is illustrated in FIGS. 59 and 60 by showing the beamfilling the objective lens aperture, but it may be applied to any systemaperture.

With the solution described above, the values FT1 and FT2 vary with thewavelength of the electromagnetic radiation used. This causes theoverall length of the telecentric relay zoom compensator to vary, asshown in FIG. 61. In FIG. 61, the length from the output of the last AODgrating to the entrance to the microscope is illustrated. As will beapparent from FIG. 61, the overall length will be equal to 2×FT1 plus2×FT2.

Accordingly, to implement this system it would be necessary to move theobjective lens relative to the AODs each time the laser wavelength ischanged. While this is feasible, it would be convenient to provide atelecentric relay that is of constant length whatever the wavelength oflight used.

One solution is to use four mirrors in a standard “optical trombone”arrangement before, or preferably after, the zoom compensator in orderto make the overall path length constant.

Another possibility is to design a system such that the sum of FT1 andFT2 is constant. This can be done by varying R with the wavelength.Since varying R will vary C (the compensation factor), s3 can be variedin order to balance this to keep C on target. C will be constant if theproduct of R and s3 is constant.

The above description of this zoom compensator has several specificaspects which should not be taken as limitations to our claims. Firstly,the example shows that the diameter of the input aperture (FIG. 61) isequal to the diameter of the output aperture. This could simply bechanged by adjusting the focal lengths of FT1 and FT2. Secondly, thefocal lengths of the sub components 115,116, 155 and 166 of eachcompensator plate 111, 151 have been taken as equal to one another inmagnitude at the design mid wavelength. The system could however bedesigned with different focal lengths here, the key point being that theresulting overall system balances the positive and negative chromaticcompensation in the overall telecentric relay so that the magnificationchromatic aberration is reduced throughout the working field, preferablywith the zero aberration point still in the middle so that the edge offield aberrations are minimised.

Accordingly, the present invention proposes the use of zoom lens systemsto adjust the overall magnification of the chromatic aberrationcorrection system so as to achieve a full objective lens aperture evenat different wavelengths.

1. Apparatus for selectively deflecting a beam of electromagneticradiation, said apparatus comprising: a first acousto-optic deflector; asecond acousto-optic deflector; a first polariser between said first andsecond acousto-optic deflectors; a first phase plate between said firstand second acousto-optic deflectors; wherein said first polariser andsaid first phase plate are arranged to pass first order components ofdiffraction and to block any zeroth order components of diffraction. 2.Apparatus according to claim 1, wherein said first acousto-opticdeflector comprises a crystal cut with less than 3 degrees deliberatemisorientation of the optic axis from the direction of propagation ofthe acoustic wave.
 3. Apparatus according to claim 1, wherein thediffracted and zeroth order electromagnetic radiation emerging from saidfirst acousto-optic deflector is elliptically polarised.
 4. Apparatusaccording to claim 1, wherein said first phase plate is selected from agroup comprising a quarter wave plate and a phase plate with a smallerfraction of wave correction.
 5. Apparatus according to claim 1, whereinsaid first and second acousto-optic deflectors are arranged to focussaid beam in a first direction.
 6. Apparatus according to claim 1,further comprising a second polariser and a second phase plate aftersaid second acousto-optic deflector, wherein said second polariser andsaid second phase plate are arranged to pass first order components ofdiffraction and to block any zeroth order components of diffraction. 7.Apparatus according to claim 1, further comprising: a thirdacousto-optic deflector; and a fourth acousto-optic deflector. 8.Apparatus according to claim 7, wherein said third and fourthacousto-optic deflectors are arranged to focus said beam in a seconddirection.
 9. Apparatus according to claim 7, wherein said acousto-opticdeflectors are arranged in this order along the path of the beam: first,third, second, fourth.
 10. An apparatus according to claim 1, whereinsaid acousto-optic deflectors are made from a high efficiencyanisotropic acousto-optic crystal.
 11. An apparatus according to claim1, wherein said acoustic-optic deflectors are made from TeO2 crystals.12. A method for selectively deflecting a beam of electromagneticradiation, said method comprising: passing said beam through a firstacousto-optic deflector; passing said beam through a first polariser anda first phase plate so as to pass the first order components ofdiffraction and to block any zeroth order components of diffraction;passing said beam through a second acousto-optic deflector.
 13. A methodaccording to claim 12, wherein said first acousto-optic deflectorcomprises a crystal cut with less than 3 degrees deliberatemisorientation of the optic axis from the direction of propagation ofthe acoustic wave.
 14. A method according to claim 12, wherein thediffracted and zeroth order electromagnetic radiation emerging from saidfirst acousto-optic deflector is elliptically polarised.
 15. A methodaccording to claim 12, wherein said first phase plate is selected from agroup comprising a quarter wave plate and a phase plate with a smallerfraction of wave correction.
 16. A method according to claim 12, whereinsaid first and second acousto-optic deflectors focus said beam in afirst direction.
 17. A method according to claim 12, further comprising:passing said beam through a second polariser and a second phase plateafter said second acousto-optic deflector so as to pass first ordercomponents of diffraction and to block any zeroth order components ofdiffraction.
 18. A method according to claim 13, further comprising:passing said beam through a third acousto-optic deflector; and passingsaid beam through a fourth acousto-optic deflector.
 19. A methodaccording to claim 18, wherein said third and fourth acousto-opticdeflectors focus said beam in a second direction.
 20. A method accordingto claim 18, wherein said acousto-optic deflectors are arranged in thisorder along the path of the beam: first, third, second, fourth.